Endothelial HIF2α suppresses retinal angiogenesis in neonatal mice by upregulating NOTCH signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

Matrix stiffness regulates Notch signaling activity in endothelial cells.

Notch signaling is critical for many developmental and disease-related processes. It is widely accepted that Notch has a mechanotransduction module that regulates receptor cleavage. However, the role of biomechanical properties of the cellular environment in Notch signaling in general is still poorly understood. During angiogenesis, differentiation of endothelial cells into tip and stalk cells is regulated by Notch signaling, and remodeling of the extracellular matrix occurs. We investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness-tuned polydimethylsiloxane (PDMS) substrates, we show that activity of the Notch signaling pathway inversely correlates with a physiologically relevant range of substrate stiffness (i.e. increased Notch signaling activity on softer substrates). Trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness, and integrin cell-matrix connections are both stiffness dependent and influenced by Notch signaling. We conclude that mechanotransduction of Notch activation is modulated by substrate stiffness, highlighting the role of substrate rigidity as an important cue for signaling. This might have implications in pathological situations associated with stiffening of the extracellular matrix, such as tumor growth.

FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Notch signaling determines cell-fate specification of the two main types of vomeronasal neurons of rodents.

The ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4  regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

Notch ligands are biomarkers of anti-TNF response in RA patients.

Notch and its ligands play a critical role in rheumatoid arthritis (RA) pathogenesis. Hence, studies were conducted to delineate the functional significance of the Notch pathway in RA synovial tissue (ST) cells and the influence of RA therapies on their expression. Morphological studies reveal that JAG1, DLL4, and Notch1 are highly enriched in RA ST lining and sublining CD68+CD14+ MΦs. JAG1 and DLL4 transcription is jointly upregulated in RA MΦs reprogrammed by TLR4/5 ligation and TNF, whereas Syntenin-1 exposure expands JAG1, DLL4, and Notch1 expression levels in these cells. Single-cell RNA-seq data exhibit that JAG1 and Notch3 are overexpressed on all fibroblast-like synoviocyte (FLS) subpopulations, in parallel, JAG2, DLL1, and Notch1 expression levels are modest on RA FLS and are predominately potentiated by TLR4 ligation. Intriguingly, JAG1, DLL1/4, and Notch1/3 are presented on RA endothelial cells, and their expression is mutually reconfigured by TLR4/5 ligation in the endothelium. Synovial JAG1/JAG2/DLL1 or Notch1/3 transcriptomes were unchanged in patients who received disease-modifying anti-rheumatic drugs (DMARDs) or IL-6R Ab therapy regardless of disease activity score. Uniquely, RA MΦs and endothelial cells rewired by IL-6 displayed DLL4 transcriptional upregulation, and IL-6R antibody treatment disrupted RA ST DLL4 transcription in good responders compared to non-responders or moderate responders. Nevertheless, the JAG1/JAG2/DLL1/DLL4 transcriptome was diminished in anti-TNF good responders with myeloid pathotype and was unaltered in the fibroid pathotype except for DLL4. Taken together, our findings suggest that RA myeloid Notch ligands can serve as markers for anti-TNF responsiveness and trans-activate Notch receptors expressed on RA FLS and/or endothelial cells.

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

Assessing angiogenesis factors as prognostic biomarkers in breast cancer patients and their association with clinicopathological factors.

INTRODUCTION: Angiogenesis is fundamental for tumor growth and metastasis across many solid malignancies. Considerable interest has focused on the molecular regulation of tumor angiogenesis as a means to predict disease outcomes and guide therapeutic decisions. METHODS: In the present study, we investigated the prognostic value of transforming growth factor beta (TGF-ß), epidermal growth factor (EGF), fibroblast growth factor (FGF), delta-like ligand 4 (DLL4), and vascular endothelial growth factor (VEGF) in the serum of 120 women diagnosed with breast cancer using ELISA as well as examined their associations with clinical parameters and the outcome of the disease. RESULTS: Our results demonstrated that the serum concentration of TGF-ß and EGF were remarkably higher in patients with higher tumor size, end stages of the disease, and positive lymph node involvement compared to patients with lower tumor size, early stages of the disease, and negative lymph node involvement. In addition, we found a significant correlation between the serum concentration of VEGF and the level of EGF, FGF, and DLL4 in patients with breast cancer. Furthermore, both univariate and multivariate analyses showed that TGF-ß and EGF can be used as end-stage predictors. DISCUSSION/CONCLUSION: Based on our findings, increasing the level of angiogenesis factors is significantly associated with higher tumor size and late stages of the disease in patients with breast cancer. Moreover, measuring the level of angiogenesis factors could lead to better prediction of disease outcomes and choosing the best treatments for patients.

COMPANION-002 A clinical trial of investigational drug CTX-009 plus paclitaxel vs paclitaxel in second line advanced BTC.

Treatment options for patients with biliary tract cancer are limited, and the prognosis is poor. CTX-009, a novel bispecific antibody targeting both DLL4 and VEGF-A, has demonstrated antitumor activity in patients with advanced cancers as both a monotherapy and in combination with chemotherapy. In a phase II study of patients with advanced biliary tract cancer who had received one or two prior therapies, CTX-009 with paclitaxel demonstrated a 37.5% overall response rate (ORR). Described here is the design of and rationale for COMPANION-002, a randomized phase II/III study, which will evaluate the safety and efficacy of CTX-009 in combination with paclitaxel versus paclitaxel alone as second-line treatment for patients with advanced biliary tract cancer. The primary end point is ORR, and crossover is allowed.Clinical Trial Registration: NCT05506943 (ClinicalTrials.gov).

Looking for new options for patients with advanced biliary tract cancer? Explore COMPANION-002, Compass Therapeutics' phase II/III study of CTX-009 + paclitaxel as a second line treatment.#CMPX #biotech #healthcare #rarecancer.

Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy.

Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.

Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice.

BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.

MEF2 transcription factors are key regulators of sprouting angiogenesis.

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

DLL4/Notch blockade disrupts mandibular advancement-induced condylar osteogenesis by inhibiting H-type angiogenesis.

BACKGROUND: Blocking Delta-like 4 (DLL4)/Notch has emerged as a promising therapeutic target for the treatment of tumours by deregulating angiogenesis. However, DLL4/Notch serves as a negative regulator of angiogenesis in multiple organs while acting as a positive regulator of H-type angiogenesis in postnatal long bones. Therefore, the effect of DLL4/Notch signalling blockade on mandibular condylar osteogenesis attracted our attention. OBJECTIVE: To explore the effect of blocking DLL4/Notch on mandibular advancement (MA)-induced condylar osteogenesis. METHODS: Six-week-old young male C57BL/6J mice (n = 40) were randomly divided into four groups: control group, MA group, MA + Anti-DLL4 group and MA + IgG group. Of note, IgG served as the isotype control for the anti-DLL4. The femurs, tibias and mandibular condyles were collected after sacrificing mice on Day 31 for morphology, micro-computed tomography, immunofluorescence, histology and immunohistochemistry evaluation. RESULTS: First, DLL4/Notch blockade shortened femoral length and reduced bone mass by inhibiting H-type angiogenesis. Second, DLL4/Notch blockade disrupted MA-induced condylar head volume and quality by inhibiting H-type angiogenesis. Mechanistically, blocking DLL4/Notch reduced the number of runt-related transcription factor 2+ (RUNX2+ ) early osteoprogenitors and the expression of Noggin protein in the condylar subchondral bone by inhibiting H-type angiogenesis. In addition, blockade of DLL4/Notch also destroyed the condylar cartilage layer. CONCLUSION: DLL4/Notch blockade results in shortened femurs and osteopenia, as well as impaired MA-induced condylar osteogenic volume and quality in growing mice by inhibiting H-type angiogenesis. Therefore, when blocking DLL4/Notch is used as a treatment target for diseases, attention should be paid to its impact on the bone mass of mandibular condyle.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

NOTCH3 and Pulmonary Arterial Hypertension.

NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted.

Novel specific anti-ESM1 antibodies overcome tumor bevacizumab resistance by suppressing angiogenesis and metastasis.

Suppressing tumors through anti-angiogenesis has been established as an effective clinical treatment strategy. Bevacizumab, a monoclonal antibody, is commonly used in various indications. However, two major challenges limit the long-term efficacy of bevacizumab: drug resistance and side effects. Bevacizumab resistance has been extensively studied at the molecular level, but no drug candidates have been developed for clinical use to overcome this resistance. In a previous study conducted by our team, a major finding was that high expression of ESM1 in bevacizumab-resistant tumors is associated with an unfavorable response to treatment. In particular, an increase in ESM1 expression contributes to heightened lung metastasis and microvascular density, which ultimately decreases the tumor's sensitivity to bevacizumab. In contrast, the silencing of ESM1 results in reduced angiogenesis and suppressed tumor growth in tumors resistant to bevacizumab. We put forward the hypothesis that targeting ESM1 could serve as a therapeutic strategy in overcoming bevacizumab resistance. In this study, a variety of anti-ESM1 antibodies with high affinity to human ESM1 were successfully prepared and characterized. Our in vivo study confirmed the establishment of a bevacizumab-resistant human colorectal cancer model and further demonstrated that the addition of anti-ESM1 monoclonal antibodies to bevacizumab treatment significantly improved tumor response while downregulating DLL4 and MMP9. In conclusion, our study suggests that anti-hESM1 monoclonal antibodies have the potential to alleviate or overcome bevacizumab resistance, thereby providing new strategies and drug candidates for clinical research in the treatment of bevacizumab-resistant colorectal cancer.

Effect of humanine on the Notch signaling pathway in myocardial infarction.

Background/aim: By applying humanin (HN) before myocardial infarction (MI), its protection in myocardial injury and the possible roles of its cellular mechanism in the Notch pathway were investigated. Materials and methods: The study was carried out at Firat University Experimental Research Center (12/24/2018-12/23/2019). Spraque-Dawley rats were divided into 10 groups: I (control) (n = 6), II (HN 6 h) (n = 6), III (HN 24 h) (n = 6), IV (HN day 7) (n = 6), V (MI 6 h) (n = 7), VI (MI 24 h) (n = 7), VII (MI day 7) (n = 7), VIII (MI+HN 6 h) (n = 7), IX (MI+HN 24 h) (n = 7), and X (MI+HN day 7) (n = 7). To create MI, 200 mg/kg of isoproterenol (ISO) was administered to the rats subcutaneously. Moreover, 252 µg/kg of HN was given intraperitoneally (ip) to the rats on its own and before MI. Molecular parameters Notch1, Notch2, Hes1, Hes2, Jagged1, Jagged2, DLL1, and DLL4 were examined using polymerase chain reaction in the heart tissue, Notch1, Hes1, and DLL4 were examined using western blot, while heart tissue was taken for histochemical examinations. Results: The mRNA expression levels of the Notch signaling members (Notch1, Notch2, Hes1, Hes2, Jagged1, Jagged2, DLL1, and DLL4) tended to decrease after MI. The Notch signaling members increased more significantly, especially toward day 7 after HN application before MI. In the western blot anylyses, the Notch1, Hes1, and DLL4 protein levels increased significantly toward day 7 in the groups given HN before MI. Moreover, the serum AST, LDH, CK-MB, and troponin I levels tended to decrease with the application of HN before MI and there was a significant decrease in edema, hemorrhage, and mononuclear cells in the heart tissue at 24 h post-MI and fibrosis on day 7 post-MI. Conclusion: HN administration before MI has a cardioprotective effect on rats via the Notch signaling pathway.

Delta-like ligand-4 regulates Notch-mediated maturation of second heart field progenitor-derived pharyngeal arterial endothelial cells.

Mesodermal progenitors in the second heart field (SHF) express Delta-like-ligand 4 (Dll4) that regulates Notch-mediated proliferation. As cells of SHF lineage mature to assume endocardial and myocardial cell fates, we have shown that Dll4 expression is lost, and the subsequent expression of another Notch ligand Jagged1 regulates Notch-mediated maturation events in the developing heart. A subset of SHF progenitors also matures to form the pharyngeal arch artery (PAA) endothelium. Dll4 was originally identified as an arterial endothelial-specific Notch ligand that plays an important role in blood vessel maturation, but its role in aortic arch maturation has not been studied to date secondary to the early lethality observed in Dll4 knockout mice. We show that, unlike in SHF-derived endocardium and myocardium, Dll4 expression persists in SHF-derived arterial endothelial cells. Using SHF-specific conditional deletion of Dll4, we demonstrate that as SHF cells transition from their progenitor state to an endothelial fate, Dll4-mediated Notch signalling switches from providing proliferative to maturation cues. Dll4 expression maintains arterial identity in the PAAs and plays a critical role in the maturation and re-organization of the 4th pharyngeal arch artery, in particular. Haploinsufficiency of Dll4 in SHF leads to highly penetrant aortic arch artery abnormalities, similar to those observed in the clinic, primarily resulting from aberrant reorganization of bilateral 4th pharyngeal arch arteries. Hence, we show that cells of SHF lineage that assume an arterial endothelial fate continue to express Dll4 and the resulting Dll4-mediated Notch signalling transitions from an early proliferative to a later maturation role during aortic arch development.

Immobilized DLL4-induced Notch signaling is mediated by dynamics of the actin cytoskeleton.

Notch signaling, which is essential for tissue development and homeostasis, has received attention as an attractive target for cancer therapy, tissue engineering and regenerative medicine. For signal activation, the Notch receptor undergoes proteolysis after binding to its ligand. This process is mediated by a mechanical pulling force, and receptor trans-endocytosis is known to play a central role in supplying the force. On the other hand, Notch ligands immobilized on carrier materials also induce artificial Notch activation. However, the mechanism of signal activation by immobilized ligand proteins is not fully understood. Here, we found that the actin cytoskeleton in Notch1-expressing cells contributes to signal activation induced by immobilized DLL4 (Delta-like ligand 4), and the results showed that pharmacological inhibition of actin dynamics impaired Notch signaling induced by DLL4-coated beads. Moreover, inhibition of actin dynamics remarkably impaired cell migration and was correlated with Notch signaling activity. We also investigated the contribution of Notch cis-endocytosis (the endocytosis of Notch receptor into signal-receiving cells) as an actin-mediated cell biological process to further explore the mechanism of Notch activation by DLL4-coated beads. Compromising the receptor cis-endocytosis pathway with the dynamin inhibitor did not alter DLL4-coated bead-induced Notch signaling, indicating that signal activation is not mediated by dynamin-dependent receptor cis-endocytosis. These findings suggest that Notch activation by immobilized ligands is primarily driven by actin-based cell movement, which might supply a sufficient mechanical force for receptor cleavage, but not by receptor cis-endocytosis.

Development of anti DLL4 Nanobody fused to truncated form of Pseudomonas exotoxin: As a novel immunotoxin to inhibit of cell proliferation and neovascularization.

Targeted tumor therapy is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) is overexpressed in tumor vasculature and plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. Immunotoxins due to their superior cell-killing ability and the relative simplicity of their preparation, have great potential in the clinical treatment of cancer. The aim of this study was to develop a novel immunotoxin against DLL4 as a cell cytotoxic agent and angiogenesis maturation inhibitor. In present study, an immunotoxin, named DLL4Nb-PE, in which a Nanobody as targeting moiety fused to the Pseudomonas exotoxin A (PE) was constructed, expressed and assessed by SDS-PAGE, western blotting, ELISA and flowcytometry. The functional assessment was carried out via MTT, apoptosis and chicken chorioallantoic membrane (CAM) assays. It was demonstrated DLL4Nb-PE specifically binds to DLL4 and recognizes DLL4-expressing MKN cells. The cytotoxicity assays showed that this molecule could induce apoptosis and kill DLL4 positive MKN cells. In addition, it inhibited neovascularization in the chicken chorioallantoic membrane. Our findings indicate designed anti-DLL4 immunotoxin has valuable potential for application to the treatment of tumors with high DLL4 expression.