Staminal hairs increase pollinator attraction and pollination accuracy in Tradescantia fluminensis (Commelinaceae).

Staminal hairs are the particular appendages of stamens, which may affect pollinator foraging behaviour and pollen transfer. However, experimental evidence of the functions of staminal hairs in pollination remains scarce. Here, we conducted staminal hair manipulation experiments in Tradescantia fluminensis (Commelinaceae) to investigate their effects on visitation and pollen transfer by bees. Our observations revealed that both visitation rates and visit duration of honeybees (Apis cerana) to control flowers were significantly higher than that of hairless flowers. Moreover, removing the staminal hairs significantly decreased pollen deposition by honeybees (A. cerana), but did not affect pollen removal. The staminal hair was similar in length to the stamen and the pistil of T. fluminensis. The staminal hairs provide more footholds for honeybees, and they lay prone on the staminal hairs to collect pollen, which increased the accuracy of pollination through the consistent pollen placement and pick-up on the ventral surface of honeybees. These results showed that the staminal hairs in T. fluminensis may represent an adaptation to attract pollinators and enhance pollination accuracy.

Microbial carbohydrate-active enzymes influence soil carbon by regulating the of plant- and fungal-derived biomass decomposition in plateau peat wetlands under differing water conditions.

Peatlands are important carbon sinks and water sources in terrestrial ecosystems. It is important to explore their microbial-driven water-carbon synergistic mechanisms to understand the driving mechanisms of carbon processes in peatlands. Based on macrogenomic sequencing techniques, located on the peatland of the eastern margin of the Tibetan Plateau with similar stand and different water conditions, we taken soil properties, microbiome abundance, CAZyme abundance and enzyme gene pathways as the object of study, investigated the characterization of soil microbial carbohydrate-active enzymes (CAZymes) under different water gradients in peatland. According to the results, these three phyla (Chloroflexi, Gemmatimonadetes, and Verrucomicrobia) differed significantly between water gradients. Under dried wetlands, the abundance of CAZymes involved in hemicellulose and glucan degradation increased by 3.0 × 10-5 and 3.0 × 10-6, respectively. In contrast, the abundance of CAZymes involved in chitin degradation decreased by 1.1 × 10-5 (p < 0.05). It highlights that regulating plant- and fungus-derived carbon metabolism processes by soil microorganisms in highland peatlands is a crucial mechanism for their response to water changes. Most plant-derived carbon fractions are regulated by soil enzymes (endo-beta 1,4-xylanase, alpha-L-arabinofuranosidase, and alpha-L-fucosidase) containing CAZymes functional genes. Additional findings in this enzyme gene pathway indicate that water changes that affect soil carbon fractions indirectly influence the three enzyme gene metabolic pathways related to plant carbon sources (the glycolysis/gluconeogenesis, other glycan degradation and amino sugar, and nucleotide sugar metabolism). Overall, this study highlights the significance of microbial CAZymes in highland peatland soil carbon processes and indicates that microbial conversion of plant and fungal biomass carbon is more sensitive to water changes.

TMEM215 Prevents Endothelial Cell Apoptosis in Vessel Regression by Blunting BIK-Regulated ER-to-Mitochondrial Ca Influx.

BACKGROUND: In developmental and pathological tissues, nascent vessel networks generated by angiogenesis require further pruning/regression to delete nonfunctional endothelial cells (ECs) by apoptosis and migration. Mechanisms underlying EC apoptosis during vessel pruning remain elusive. TMEM215 (transmembrane protein 215) is an endoplasmic reticulum-located, 2-pass transmembrane protein. We have previously demonstrated that TMEM215 knockdown in ECs leads to cell death, but its physiological function and mechanism are unclear. METHODS: We characterized the role and mechanism of TMEM215 in EC apoptosis using human umbilical vein endothelial cells by identifying its interacting proteins with immunoprecipitation-mass spectrometry. The physiological function of TMEM215 in ECs was assessed by establishing a conditional knockout mouse strain. The role of TMEM215 in pathological angiogenesis was evaluated by tumor and choroidal neovascularization models. We also tried to evaluate its translational value by delivering a Tmem215 small interfering RNA (siRNA) using nanoparticles in vivo. RESULTS: TMEM215 knockdown in ECs induced apoptotic cell death. We identified the chaperone BiP as a binding partner of TMEM215, and TMEM215 forms a complex with and facilitates the interaction of BiP (binding immunoglobin protein) with the BH (BCL-2 [B-cell lymphoma 2] homology) 3-only proapoptotic protein BIK (BCL-2 interacting killer). TMEM215 knockdown triggered apoptosis in a BIK-dependent way and was abrogated by BCL-2. Notably, TMEM215 knockdown increased the number and diminished the distance of mitochondria-associated endoplasmic reticulum membranes and increased mitochondrial calcium influx. Inhibiting mitochondrial calcium influx by blocking the IP3R (inositol 1,4,5-trisphosphate receptor) or MCU (mitochondrial calcium uniporter) abrogated TMEM215 knockdown-induced apoptosis. TMEM215 expression in ECs was induced by physiological laminar shear stress via EZH2 downregulation. In EC-specific Tmem215 knockout mice, induced Tmem215 depletion impaired the regression of retinal vasculature characterized by reduced vessel density, increased empty basement membrane sleeves, and increased EC apoptosis. Moreover, EC-specific Tmem215 ablation inhibited tumor growth with disrupted vasculature. However, Tmem215 ablation in adult mice attenuated lung metastasis, consistent with reduced Vcam1 expression. Administration of nanoparticles carrying Tmem215 siRNA also inhibited tumor growth and choroidal neovascularization injury. CONCLUSIONS: TMEM215, which is induced by blood flow-derived shear stress via downregulating EZH2, protects ECs from BIK-triggered mitochondrial apoptosis mediated by calcium influx through mitochondria-associated ER membranes during vessel pruning, thus providing a novel target for antiangiogenic therapy.

Repression of rRNA gene transcription by endothelial SPEN deficiency normalizes tumor vasculature via nucleolar stress.

Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and blunts most current therapies; however, the mechanisms underlying the induction of a dysfunctional vasculature have been unclear. Here, we show that split end (SPEN), a transcription repressor, coordinates rRNA synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, a low endothelial SPEN level correlated with extended overall survival. In mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, and this was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization and combining CX-5461 with cisplatin substantially improved the efficacy of treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis and that RNPI represents a target for tumor vessel normalization therapy of cancer.

miR-342-5p promotes vascular smooth muscle cell phenotypic transition through a negative-feedback regulation of Notch signaling via targeting FOXO3.

AIM: Under various pathological conditions such as cancer, vascular smooth muscle cells (vSMCs) transit their contractile phenotype into phenotype(s) characterized by proliferation and secretion, a process called vSMC phenotypic transition (vSMC-PT). Notch signaling regulates vSMC development and vSMC-PT. This study aims to elucidate how the Notch signal is regulated. MAIN METHODS: Gene-modified mice with a SM22α-CreERT2 transgene were generated to activate/block Notch signaling in vSMCs. Primary vSMCs and MOVAS cells were cultured in vitro. RNA-seq, qRT-PCR and Western blotting were used to evaluated gene expression level. EdU incorporation, Transwell and collagen gel contraction assays were conducted to determine the proliferation, migration and contraction, respectively. KEY FINDINGS: Notch activation upregulated, while Notch blockade downregulated, miR-342-5p and its host gene Evl in vSMCs. However, miR-342-5p overexpression promoted vSMC-PT as shown by altered gene expression profile, increased migration and proliferation, and decreased contraction, while miR-342-5p blockade exhibited the opposite effects. Moreover, miR-342-5p overexpression significantly suppressed Notch signaling, and Notch activation partially abolished miR-342-5p-induced vSMC-PT. Mechanically, miR-342-5p directly targeted FOXO3, and FOXO3 overexpression rescued miR-342-5p-induced Notch repression and vSMC-PT. In a simulated tumor microenvironment, miR-342-5p was upregulated by tumor cell-derived conditional medium (TCM), and miR-342-5p blockade abrogated TCM-induced vSMC-PT. Meanwhile, conditional medium from miR-342-5p-overexpressing vSMCs significantly enhanced tumor cell proliferation, while miR-342-5p blockade had the opposite effects. Consistently, in a co-inoculation tumor model, miR-342-5p blockade in vSMCs significantly delayed tumor growth. SIGNIFICANCE: miR-342-5p promotes vSMC-PT through a negative-feedback regulation of Notch signaling via downregulating FOXO3, which could be a potential target for cancer therapy.

The Notch signaling pathway: a potential target for cancer immunotherapy.

Dysregulation of the Notch signaling pathway, which is highly conserved across species, can drive aberrant epigenetic modification, transcription, and translation. Defective gene regulation caused by dysregulated Notch signaling often affects networks controlling oncogenesis and tumor progression. Meanwhile, Notch signaling can modulate immune cells involved in anti- or pro-tumor responses and tumor immunogenicity. A comprehensive understanding of these processes can help with designing new drugs that target Notch signaling, thereby enhancing the effects of cancer immunotherapy. Here, we provide an up-to-date and comprehensive overview of how Notch signaling intrinsically regulates immune cells and how alterations in Notch signaling in tumor cells or stromal cells extrinsically regulate immune responses in the tumor microenvironment (TME). We also discuss the potential role of Notch signaling in tumor immunity mediated by gut microbiota. Finally, we propose strategies for targeting Notch signaling in cancer immunotherapy. These include oncolytic virotherapy combined with inhibition of Notch signaling, nanoparticles (NPs) loaded with Notch signaling regulators to specifically target tumor-associated macrophages (TAMs) to repolarize their functions and remodel the TME, combining specific and efficient inhibitors or activators of Notch signaling with immune checkpoint blockers (ICBs) for synergistic anti-tumor therapy, and implementing a customized and effective synNotch circuit system to enhance safety of chimeric antigen receptor (CAR) immune cells. Collectively, this review aims to summarize how Notch signaling intrinsically and extrinsically shapes immune responses to improve immunotherapy.

miR-342-5p downstream to Notch enhances arterialization of endothelial cells in response to shear stress by repressing MYC.

During vascular development, endothelial cells (ECs) undergo arterialization in response to genetic programs and shear stress-triggered mechanotransduction, forming a stable vasculature. Although the Notch receptor is known to sense shear stress and promote EC arterialization, its downstream mechanisms remain unclear. In this study, the Notch downstream miR-342-5p was found to respond to shear stress and promote EC arterialization. Shear stress upregulated miR-342-5p in a Notch-dependent manner in human umbilical vein endothelial cells (HUVECs). miR-342-5p overexpression upregulated the shear stress-associated transcriptomic signature. Moreover, miR-342-5p upregulated arterial markers and promoted EC arterialization in a Matrigel plug assay and retinal angiogenesis model. In contrast, miR-342-5p knockdown downregulated arterial markers, compromised retinal arterialization, and partially abrogated shear stress and Notch activation-induced arterial marker upregulation. Mechanistically, miR-342-5p overexpression suppressed MYC to repress EC proliferation and promote arterialization, achieved by promoting MYC protein degradation by targeting the EYA3. Consistently, EYA3 overexpression rescued miR-342-5p-mediated MYC downregulation and EC arterialization. In vivo, miR-342-5p expression was notably decreased in the ligated artery in a hindlimb ischemia model, and an intramuscular injection of miR-342-5p promoted EC arterialization and improved perfusion. In summary, miR-342-5p, a mechano-miR, mediates the effects of shear stress-activated Notch on EC arterialization and is a potential therapeutic target for ischemic diseases.

Research Progress on the Pathogenesis of Aortic Dissection.

Aortic dissection is a critical cardiovascular disease due to the separation of media and adventitia caused by the rupture of vascular wall intima. The disease has a high mortality rate of about 1%-3% for each additional hour, since the adventitia of the aorta can rupture and bleed to death at any time. Although great progress has been made in clinical treatment of aortic dissection, and the mortality rate has been significantly reduced, the pathogenesis is still not very clear. At present, related studies have confirmed that inflammation of aortic wall promotes the occurrence and development of Aortic dissection. Although the mechanism of aortic dissection is more complicated, some studies have shown that the infiltration of monocytes/macrophages into the aortic wall is the main pathogenic mechanism of the disease. This review introduces the latest research results on the mechanism of macrophage infiltration and plasticity in aortic dissection.

RNA-binding protein SPEN controls hepatocyte maturation via regulating Hnf4α expression during liver development.

Liver organogenesis is a complex process. Although many signaling pathways and key factors have been identified during liver development, little is known about the regulation of late liver development, especially liver maturation. As a transcriptional repressor, SPEN has been demonstrated to interact with lncRNAs and transcription factors to participate in X chromosome inactivation, neural development, and lymphocyte differentiation. General disruption of SPEN results in embryonic lethality accompanied by hampered liver development in mice. However, the function of SPEN in embryonic liver development has not been reported. In this study, we demonstrate that SPEN is required for hepatocyte maturation using hepatocyte-specific disruption of SPEN with albumin-Cre-mediated knockout. SPEN expression was upregulated in hepatocytes along with liver development in mice. The deletion of the SPEN gene repressed hepatic maturation, mainly by a decrease in hepatic metabolic function and disruption of hepatocyte zonation. Additional experiments revealed that transcription factors which control hepatocyte maturation were strongly downregulated in SPEN-deficient hepatocytes, especially Hnf4α. Furthermore, restoration of Hnf4α levels partially rescued the immature state of hepatocytes caused by SPEN gene deletion. Taken together, these results reveal an unexpected role of SPEN in liver maturation.

Persistent calyces increase floral longevity and female fitness in Salvia miltiorrhiza (Lamiaceae).

The evolution of persistent calyces may be an adaptation to ensure reproductive success of certain flowering plants. However, experimental evidence of the functions of persistent calyces during flowering and seed development remains scarce. We explored the possible functions of persistent calyces in Salvia miltiorrhiza, a perennial herb with campanulate calyx. We conducted calyx manipulation experiments to examine whether persistent calyces affect visitation rates of nectar robbers and pollinators, individual flower longevity, fruit set, seed set and seed mass. Our findings suggested that shortening of the calyx significantly decreased individual flower longevity, fruit set and seed mass, but did not affect visitation of pollinators and nectar robbers. In addition, the seed set of control flowers and the flowers with calyx shortened at the beginning of fruiting stage (CSF flowers) did not significantly differ, but both were higher than that of the flowers with calyx shortened at the beginning of blooming stage (CSB flowers). The seed set and fruit set of CSB flowers were limited by pollination due to the reduction in floral longevity. We conclude that persistent calyces of S. miltiorrhiza may represent adaptive strategies to maintain floral longevity and increase plant fitness. Persistent calyces may provide protection for the growth of flowers and contribute resources to the development of fruits and seeds.

Notch activation suppresses endothelial cell migration and sprouting via miR-223-3p targeting Fbxw7.

Angiogenesis involves temporo-spatially coordinated endothelial cell (EC) proliferation, differentiation, migration, and sprouting. Notch signaling is essential in regulating EC behaviors during angiogenesis, but its downstream mechanisms remain incompletely defined. In the current study, we show that miR-223-3p is a downstream molecule of Notch signaling and mediates the role of Notch signaling in regulating EC migration and sprouting. In human umbilical vein endothelial cells (HUVECs), Notch activation by immobilized Dll4, a Notch ligand, upregulated miR-223-3p, and Notch activation-mediated miR-223-3p upregulation could be blocked by a γ-secretase inhibitor (DAPT). miR-223-3p overexpression apparently repressed HUVEC migration, leading to attenuated lumen formation and sprouting capacities. Transcriptome comparison and subsequent qRT-PCR validation further indicated that miR-223-3p downregulated the expression of multiple genes involved in EC migration, axon guidance, extracellular matrix remodeling, and angiogenesis. In addition, miR-223-3p antagonist transfection abolished Notch-mediated repression of EC migration and sprouting. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting, and reporter assay analysis, we confirmed that miR-223-3p directly targeted F-box and WD repeat domain-containing 7 (Fbxw7). Meanwhile, Fbxw7 overexpression could efficiently rescue the impaired migration capacity of ECs under miR-223-3p overexpression. In summary, these results identify that Notch activation-induced miR-223-3p suppresses EC migration and sprouting via Fbxw7.

Prevalence and genotype distribution of human papillomavirus infection among women aged 30-65 years in Xi'an, China: a population-based study of 14,655 women.

Cervical cancer is the fourth most common cancer among women worldwide in terms of both incidence and mortality. Persistent infection with high-risk human papillomavirus (HPV) has been identified as a cause of cervical intraepithelial neoplasia and invasive cervical cancer. The distribution of human papillomavirus genotypes varies regionally. To acquire baseline data on the population-based prevalence and genotype distribution of HPV infection, we investigated the molecular epidemiology of HPV infection among women in Xi'an, China. The study was conducted from September 2018 to December 2020. A total of 14,655 women aged 30-65 years were screened. The overall prevalence of HPV infection was 13.5% (95% confidence interval [CI]: 13.0-14.1%), with 10.4% of participants being positive for a single HPV type and 3.1% being positive for multiple HPV types. The prevalence of high-risk HPV (HR-HPV), low-risk HPV (LR-HPV) and mixed HPV infection was 10.1% (95% CI: 9.6-10.5%), 2.2% (95% CI: 2.0-2.4%), and 1.3% (95% CI: 1.1-1.5%), respectively. The five most frequently detected HR-HPV types were types 52 (2.6%), 16 (1.9%), 53 (1.8%), 58 (1.4%), and 51 (0.9%). The most frequently detected LR-HPV type was HPV-42 (1.1%). The prevalence and HPV genotype distribution varied by region and age. Age-specific HPV prevalence peaked in the over 60 years age group (18.8%), and Beilin District had the highest HPV prevalence (18.1%). The results of this first population-based study provide a reference for HPV-based cervical cancer screening and HPV vaccination programs in Xi'an.

Notch activation promotes endothelial quiescence by repressing MYC expression via miR-218.

After angiogenesis-activated embryonic and early postnatal vascularization, endothelial cells (ECs) in most tissues enter a quiescent state necessary for proper tissue perfusion and EC functions. Notch signaling is essential for maintaining EC quiescence, but the mechanisms of action remain elusive. Here, we show that microRNA-218 (miR-218) is a downstream effector of Notch in quiescent ECs. Notch activation upregulated, while Notch blockade downregulated, miR-218 and its host gene Slit2, likely via transactivation of the Slit2 promoter. Overexpressing miR-218 in human umbilical vein ECs (HUVECs) significantly repressed cell proliferation and sprouting in vitro. Transcriptomics showed that miR-218 overexpression attenuated the MYC proto-oncogene, bHLH transcription factor (MYC, also known as c-myc) signature. MYC overexpression rescued miR-218-mediated proliferation and sprouting defects in HUVECs. MYC was repressed by miR-218 via multiple mechanisms, including reduction of MYC mRNA, repression of MYC translation by targeting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and promoting MYC degradation by targeting EYA3. Inhibition of miR-218 partially reversed Notch-induced repression of HUVEC proliferation and sprouting. In vivo, intravitreal injection of miR-218 reduced retinal EC proliferation accompanied by MYC repression, attenuated pathological choroidal neovascularization, and rescued retinal EC hyper-sprouting induced by Notch blockade. In summary, miR-218 mediates the effect of Notch activation of EC quiescence via MYC and is a potential treatment for angiogenesis-related diseases.

Do flowers removed of either nectar or pollen attract fewer bumblebee pollinators? An experimental test in Impatiens oxyanthera.

Pollen and nectar are the primary rewards offered by flowers to pollinators. In floral visitors of some plant species, pollen thieves and nectar robbers cause the reduction in pollen grain number and nectar volume, respectively. However, it remains unclear whether the absence of either of the two rewards in a given flower reduces its attraction to nectar- and pollen-collecting pollinators. We hypothesized that flowers removed of either nectar or pollen would attract fewer pollinators. We studied protandrous Impatiens oxyanthera, whose flowers provide bumblebee pollinators with both nectar and pollen in the male phase. We conducted floral reward manipulation experiments to explore how the removal of either nectar or pollen from flowers influences pollinator behaviour by comparing their visitation rates and visit duration. Compared with the control flowers, the flowers removed of pollen attracted significantly more bumblebee pollinators per 30 min, but the flowers removed of nectar or those removed of both pollen and nectar attracted significantly fewer bumblebee pollinators per 30 min. Moreover, the visit duration of bumblebee pollinators to control flowers or flowers removed of pollen was longer than that to flowers removed of nectar or those removed of both pollen and nectar. Our investigations indicated that compared with control flowers, the flowers removed of nectar attracted fewer bumblebee pollinators, supporting our hypothesis. However, our other hypothesis that pollen removal would reduce pollinator visits was not supported by our results. Instead, compared with control flowers, the flowers that contained only nectar attracted more bumblebee pollinators. Nectar seems to be the main reward, and bumblebee pollinators mainly used the absence of pollen as a visual signal to locate I. oxyanthera flowers with a potentially higher amount of nectar.

[A probe to the Empowerment and Efficiency Oriented teaching reform of molecular biology experiment].

Molecular biology theory and technology, as a frontier subject of life sciences, are developing rapidly. As the key link between theory and practice, molecular biology experimental courses play an important role in the cultivation of students' critical thinking and innovation abilities. This paper analyzed the critical issues in experimental teaching by collecting and reflecting on students' feedbacks. With a focus on the Empowerment and Efficiency Oriented Teaching, the study was aimed at improving students' hands-on and thinking ability, and raising the efficacy of education. A series of measures were taken in attempts to renew the setting of teaching content and reconstruct the classroom timing allotment. The reform of the teaching scheme not only significantly increases students' understanding and mastery of knowledge, and empowers students with stronger thinking and learning abilities, but also enhances the teaching efficacy of experimental courses and further improves students' scientific literacy.

SM22α+ vascular mural cells are essential for vessel stability in tumors and undergo phenotype transition regulated by Notch signaling.

BACKGROUND: Malformation of blood vessels represents a hallmark of cancers, but the role and regulation of vascular mural cells (vMCs), including vascular smooth muscle cells (vSMCs) and pericytes, in tumors has not been fully understood. SM22α has been identified as a marker of vSMCs. This study aims at elucidating the function and regulation of SM22α+ mural cells (SM22-MCs) in tumor stroma. METHODS: Gene-modified mice with a SM22α-CreERT2 transgene were employed to deplete SM22-MCs or activate/block Notch signaling in these cells. vSMCs from mouse dorsal aorta (vSMCs-DA) were cultured in vitro. RNA-seq was used to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence was used to observe morphological alterations in tumors. RESULTS: SM22-MCs are essential for stabilizing tumor vasculature. Notch signaling was downregulated in tumor-derived SM22-MCs and vSMCs-DA treated with cancer cell-derived conditioned medium. Notch activation in SM22-MCs normalized tumor vasculature and repressed tumor growth. On the other hand, Notch disruption aggravated abnormal tumor vasculature and promoted growth and metastasis. Gene expression profiling of vSMCs-DA showed that Notch activation enhances their contractile phenotype and suppresses their secretory phenotype, further attenuating the invasion and proliferation of tumor cells. In contrast, Notch blockade in vSMCs-DA mitigated their contractile phenotype while strengthened the secretory phenotype. CONCLUSION: SM22-MCs facilitate vessel stability in tumors, and they gain a secretory phenotype and promote tumor malignancy in the absence of Notch signaling.

Endothelial Notch activation promotes neutrophil transmigration via downregulating endomucin to aggravate hepatic ischemia/reperfusion injury.

Inflammatory leukocytes infiltration is orchestrated by mechanisms involving chemokines, selectins, addressins and other adhesion molecules derived from endothelial cells (ECs), but how they respond to inflammatory cues and coordinate leukocyte transmigration remain elusive. In this study, using hepatic ischemia/reperfusion injury (HIRI) as a model, we identified that endothelial Notch activation was rapidly and dynamically induced in liver sinusoidal endothelial cells (LSECs) in acute inflammation. In mice with EC-specific Notch activation (NICeCA), HIRI induced exacerbated liver damage. Consistently, endothelial Notch activation enhanced neutrophil infiltration and tumor necrosis factor (TNF)-α expression in HIRI. Transcriptome analysis and further qRT-PCR as well as immunofluorescence indicated that endomucin (EMCN), a negative regulator of leukocyte adhesion, was downregulated in LSECs from NICeCA mice. EMCN was downregulated during HIRI in wild-type mice and in vitro cultured ECs insulted by hypoxia/re-oxygenation injury. Notch activation in ECs led to increased neutrophil adhesion and transendothelial migration, which was abrogated by EMCN overexpression in vitro. In mice deficient of RBPj, the integrative transcription factor of canonical Notch signaling, although overwhelming sinusoidal malformation aggravated HIRI, the expression of EMCN was upregulated; and pharmaceutical Notch blockade in vitro also upregulated EMCN and inhibited transendothelial migration of neutrophils. The Notch activation-exaggerated HIRI was compromised by blocking LFA-1, which mediated leukocyte adherence by associating with EMCN. Therefore, endothelial Notch signaling controls neutrophil transmigration via EMCN to modulate acute inflammation in HIRI.

Transmembrane protein 215 promotes angiogenesis by maintaining endothelial cell survival.

Sprouting angiogenesis is a major form of neovascularization of tissues suffering from hypoxia and other related stress. Endothelial cells (ECs) undergo proliferation, differentiation, programmed death, and migration during angiogenic sprouting, but the underlying molecular mechanisms regulating ECs in angiogenesis have been incompletely elucidated. Here we report that the transmembrane protein 215 (TMEM215) is involved in angiogenesis by regulating EC survival. The murine TMEM215 gene, which possesses two transcriptional starting sites as determined by 5'-rapid amplification of complementary DNA (cDNA) ends (RACE), encodes a two-pass TMEM. The TMEM215 transcripts were detected in ECs in addition to other tissues by quantitative reverse transcription-polymerase chain reaction. Immunofluorescence showed that TMEM215 was expressed in the vasculature in retina, liver, and tumor, and colocalized with EC markers. We show that knockdown of TMEM215 in ECs induced strong cell death of ECs in vitro without affecting cell proliferation and migration, suggesting that TMEM215 was required for EC survival. Downregulation of TMEM215 expression compromised lumen formation and sprouting capacities of ECs in vitro. Moreover, intravitreous injection of TMEM215 small interfering RNA resulted in delayed and abnormal development of retinal vasculature with poor perfusion. These results identified TMEM215 as a novel molecule involved in angiogenesis by regulating the survival of ECs.

SNAI1, an endothelial-mesenchymal transition transcription factor, promotes the early phase of ocular neovascularization.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.

Endothelial Notch activation reshapes the angiocrine of sinusoidal endothelia to aggravate liver fibrosis and blunt regeneration in mice.

Liver sinusoidal endothelial cells (LSECs) critically regulate liver homeostasis and diseases through angiocrine factors. Notch is critical in endothelial cells (ECs). In the current study, Notch signaling was activated by inducible EC-specific expression of the Notch intracellular domain (NIC). We found that endothelial Notch activation damaged liver homeostasis. Notch activation resulted in decreased fenestration and increased basement membrane, and a gene expression profile with decreased LSEC-associated genes and increased continuous EC-associated genes, suggesting LSEC dedifferentiation. Consistently, endothelial Notch activation enhanced hepatic fibrosis (HF) induced by CCl4 . Notch activation attenuated endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) signaling, and activation of sGC by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) reversed the dedifferentiation phenotype. In addition, Notch activation subverted the hepatocyte-supporting angiocrine profile of LSECs by down-regulating critical hepatocyte mitogens, including Wnt2a, Wnt9b, and hepatocyte growth factor (HGF). This led to compromised hepatocyte proliferation under both quiescent and regenerating conditions. Whereas expression of Wnt2a and Wnt9b was dependent on eNOS-sGC signaling, HGF expression was not rescued by the sGC activator, suggesting heterogeneous mechanisms of LSECs to maintain hepatocyte homeostasis. CONCLUSION: Endothelial Notch activation results in LSEC dedifferentiation and accelerated liver fibrogenesis through eNOS-sGC signaling, and alters the angiocrine profile of LSECs to compromise hepatocyte proliferation and liver regeneration (LR). (Hepatology 2018).