Unveiling the role of circRBBP7 in myoblast proliferation and differentiation: A novel regulator of muscle development.

Muscle development is a multistep process regulated by diverse gene networks, and circRNAs are considered novel regulators mediating myogenesis. Here, we systematically analyzed the role and underlying regulatory mechanisms of circRBBP7 in myoblast proliferation and differentiation. Results showed that circRBBP7 has a typical circular structure and encodes a 13 -kDa protein. By performing circRBBP7 overexpression and RNA interference, we found that the function of circRBBP7 was positively correlated with the proliferation and differentiation of myoblasts. Using RNA sequencing, we identified 1633 and 532 differentially expressed genes (DEGs) during myoblast proliferation or differentiation, respectively. The DEGs were found mainly enriched in cell cycle- and skeletal muscle development-related pathways, such as the MDM2/p53 and PI3K-Akt signaling pathways. Further co-IP and IF co-localization analysis revealed that VEGFR-1 is a target of circRBBP7 in myoblasts. qRT-PCR and WB analysis further confirmed the positive correlation between VEGFR-1 and circRBBP7. Moreover, we found that in vivo transfection of circRBBP7 into injured muscle tissues significantly promoted the regeneration and repair of myofibers in mice. Therefore, we speculate that circRBBP7 may affect the activity of MDM2 by targeting VEGFR-1, altering the expression of muscle development-related genes by mediating p53 degradation, and ultimately promoting myoblast development and muscle regeneration. This study provides essential evidence that circRBBP7 can serve as a potential target for myogenesis regulation and a reference for the application of circRBBP7 in cattle genetic breeding and muscle injury treatment.

Endothelial ß-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR/HDAC9.

BACKGROUND: Diabetic angiogenesis is closely associated with disabilities and death caused by diabetic microvascular complications. Advanced glycation end products (AGEs) are abnormally accumulated in diabetic patients and are a key pathogenic factor for diabetic angiogenesis. The present study focuses on understanding the mechanisms underlying diabetic angiogenesis and identifying therapeutic targets based on these mechanisms. METHODS: In this study, AGE-induced angiogenesis serves as a model to investigate the mechanisms underlying diabetic angiogensis. Mouse aortic rings, matrigel plugs, and HUVECs or 293T cells were employed as research objects to explore this pathological process by using transcriptomics, gene promoter reporter assays, virtual screening and so on. RESULTS: Here, we found that AGEs activated Wnt/ß-catenin signaling pathway and enhanced the ß-catenin protein level by affecting the expression of ß-catenin degradation-related genes, such as FZDs (Frizzled receptors), LRPs (LDL Receptor Related Proteins), and AXIN1. AGEs could also mediate ß-catenin Y142 phosphorylation through VEGFR1 isoform5. These dual effects of AGEs elevated the nuclear translocation of ß-catenin and sequentially induced the expression of KDR (Kinase Insert Domain Receptor) and HDAC9 (Histone Deacetylase 9) by POU5F1 and NANOG, respectively, thus mediating angiogenesis. Finally, through virtual screening, Bioymifi, an inhibitor that blocks VEGFR1 isoform5-ß-catenin complex interaction and alleviates AGE-induced angiogenesis, was identified. CONCLUSION: Collectively, this study offers insight into the pathophysiological functions of ß-catenin in diabetic angiogenesis.

Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

The VEGFs/VEGFRs system in Alzheimer's and Parkinson's diseases: Pathophysiological roles and therapeutic implications.

The vascular endothelial growth factors (VEGFs) and their cognate receptors (VEGFRs), besides their well-known involvement in physiological angiogenesis/lymphangiogenesis and in diseases associated to pathological vessel formation, play multifaceted functions in the central nervous system (CNS). In addition to shaping brain development, by controlling cerebral vasculogenesis and regulating neurogenesis as well as astrocyte differentiation, the VEGFs/VEGFRs axis exerts essential functions in the adult brain both in physiological and pathological contexts. In this article, after describing the physiological VEGFs/VEGFRs functions in the CNS, we focus on the VEGFs/VEGFRs involvement in neurodegenerative diseases by reviewing the current literature on the rather complex VEGFs/VEGFRs contribution to the pathogenic mechanisms of Alzheimer's (AD) and Parkinson's (PD) diseases. Thereafter, based on the outcome of VEGFs/VEGFRs targeting in animal models of AD and PD, we discuss the factual relevance of pharmacological VEGFs/VEGFRs modulation as a novel and potential disease-modifying approach for these neurodegenerative pathologies. Specific VEGFRs targeting, aimed at selective VEGFR-1 inhibition, while preserving VEGFR-2 signal transduction, appears as a promising strategy to hit the molecular mechanisms underlying AD pathology. Moreover, therapeutic VEGFs-based approaches can be proposed for PD treatment, with the aim of fine-tuning their brain levels to amplify neurotrophic/neuroprotective effects while limiting an excessive impact on vascular permeability.

Meroterpenoids from Marine Sponge Hyrtios sp. and Their Anticancer Activity against Human Colorectal Cancer Cells.

Two new meroterpenoids, hyrtamide A (1) and hyrfarnediol A (2), along with two known ones, 3-farnesyl-4-hydroxybenzoic acid methyl ester (3) and dictyoceratin C (4), were isolated from a South China Sea sponge Hyrtios sp. Their structures were elucidated by NMR and MS data. Compounds 2-4 exhibited weak cytotoxicity against human colorectal cancer cells (HCT-116), showing IC50 values of 41.6, 45.0, and 37.3 µM, respectively. Furthermore, compounds 3 and 4 significantly suppressed the invasion of HCT-116 cells while also downregulating the expression of vascular endothelial growth factor receptor 1 (VEGFR-1) and vimentin proteins, which are key markers associated with angiogenesis and epithelial-mesenchymal transition (EMT). Our findings suggest that compounds 3 and 4 may exert their anti-invasive effects on tumor cells by inhibiting the expression of VEGFR-1 and impeding the process of EMT.

Anti-TPO-mediated specific features of the placenta immunohistochemical profile and possible mechanisms for fetal loss.

Passive transfer of antithyroid antibodies in mice leads to reproductive disorders. The purpose was to assess the placental tissue of experimental animals under the influence of the circulating thyroperoxidase antibodies. We performed an immunohistochemical examination of murine placentae after a passive transfer of thyroperoxidase antibodies. Placentae of mice that passively transferred IgG from healthy donors were used as control samples. For histological examination, 30 placental samples were selected from mice from the anti-TPO group and 40 placental samples were taken from mice from the IgG group. Immunostaining for VEGFR1, THBS 1, Laminin, CD31, CD34, FGF-ß, CD56, CD14, TNF-α, kisspeptin, MCL 1, and Annexin V was performed. There is a significant decrease in the relative area of the expression of VEGFR1 (23.42 ± 0.85 vs. 33.44 ± 0.35, P < 0.01), thrombospondin 1 (31.29 ± 0.83 vs. 34.51 ± 0.75, P < 0.01), CD14 (25.80 ± 0.57 vs. 32.07 ± 0.36, P < .01), CD56 (30.08 ± 0.90 vs. 34.92 ± 0.15, P < 0.01), kisspeptin (25.94 ± 0.47 vs. 31.27 ± 0.57, P < 0.01), MCL 1 (29.24 ± 1.06 vs. 38.57 ± 0.79, P < 0.01) in the labyrinth zone of the placentae of mice from the anti-TPO group compared with control group. A significant increase in the relative expression of laminin and FGF-ß was noted in the group of mice to which antibodies to thyroperoxidase were transferred, compared with the control group (36.73 ± 1.38 vs. 29.83 ± 0.94, P < 0.01 and 23.26 ± 0.61 vs. 16.38 ± 1.01, P < 0.01respectively). Our study exposed an imbalance of pro- and anti-angiogenic factors, decreased representation of placental macrophages and NK cells, abnormal trophoblast invasion processes, and insufficient expression of antiapoptotic factors in the placentae of mice in which anti-TPO antibodies were passively transferred.

The Role of Membrane-Type 1 Matrix Metalloproteinase-Substrate Interactions in Pathogenesis.

A protease is an enzyme with a proteolytic activity that facilitates the digestion of its substrates. Membrane-type I matrix metalloproteinase (MT1-MMP), a member of the broader matrix metalloproteinases (MMP) family, is involved in the regulation of diverse cellular activities. MT1-MMP is a very well-known enzyme as an activator of pro-MMP-2 and two collagenases, MMP-8 and MMP-13, all of which are essential for cell migration. As an anchored membrane enzyme, MT1-MMP has the ability to interact with a diverse group of molecules, including proteins that are not part of the extracellular matrix (ECM). Therefore, MT1-MMP can regulate various cellular activities not only by changing the extra-cellular environment but also by regulating cell signaling. The presence of both intracellular and extra-cellular portions of MT1-MMP can allow it to interact with proteins on both sides of the cell membrane. Here, we reviewed the MT1-MMP substrates involved in disease pathogenesis.

Serum Levels of VEGF-A and Its Receptors in Patients in Different Phases of Hemorrhagic and Ischemic Strokes.

Vascular endothelial growth factors (VEGFs) are important regulators of angiogenesis, neuroprotection, and neurogenesis. Studies have indicated the association of VEGF dysregulation with the development of neurodegenerative and cerebrovascular diseases. We studied the changes in serum levels of VEGF-A, VEGFR-1, and VEGFR-2 in patients at various phases of ischemic and hemorrhagic strokes. Quantitative assessment of VEGF-A, VEGFR-1, and VEGFR-2 in serum of patients with hemorrhagic or ischemic stroke was performed by enzyme immunoassay in the hyper-acute (1−24 h from the onset), acute (up to 1−7 days), and early subacute (7 days to 3 months) phases of stroke, and then compared with the control group and each other. Results of our retrospective study demonstrated different levels of VEGF-A and its receptors at various phases of ischemic and hemorrhagic strokes. In ischemic stroke, increased VEGFR-2 level was found in the hyper-acute (p = 0.045) and acute phases (p = 0.024), while elevated VEGF-A and reduced VEGFR-1 levels were revealed in the early subacute phase (p = 0.048 and p = 0.012, respectively). In hemorrhagic stroke, no significant changes in levels of VEGF-A and its receptors were identified in the hyper-acute phase. In the acute and early subacute phases there was an increase in levels of VEGF-A (p < 0.001 and p = 0.006, respectively) and VEGFR-2 (p < 0.001 and p = 0.012, respectively). Serum levels of VEGF-A and its receptors in patients with hemorrhagic and ischemic stroke indicate different pathogenic pathways depending on the phase of the disease.

Pericytes Regulate Cerebral Perfusion through VEGFR1 in Ischemic Stroke.

Neurons in the penumbra (the area surrounding ischemic tissue that consists of still viable tissue but with reduced blood flow and oxygen transport) may be rescued following stroke if adequate perfusion is restored in time. It has been speculated that post-stroke angiogenesis in the penumbra can reduce damage caused by ischemia. However, the mechanism for neovasculature formation in the brain remains unclear and vascular-targeted therapies for brain ischemia remain suboptimal. Here, we show that VEGFR1 was highly upregulated in pericytes after stroke. Knockdown of VEGFR1 in pericytes led to increased infarct area and compromised post-ischemia vessel formation. Furthermore, in vitro studies confirmed a critical role for pericyte-derived VEGFR1 in both endothelial tube formation and pericyte migration. Interestingly, our results show that pericyte-derived VEGFR1 has opposite effects on Akt activity in endothelial cells and pericytes. Collectively, these results indicate that pericyte-specific expression of VEGFR1 modulates ischemia-induced vessel formation and vascular integrity in the brain.

HIF1A promotes miR-210/miR-424 transcription to modulate the angiogenesis in HUVECs and HDMECs via sFLT1 under hypoxic stress.

Angiogenesis is a critical process during human skin wound healing. However, hypoxia might lead to the dysfunction of the cellular interplay of endothelial cells and subcutaneous fibroblasts, resulting in the deregulation of angiogenesis. HIF1A is a key regulatory of the recovery of intracellular homeostasis under hypoxia. In the present study, the detailed role and mechanism of HIF1A in the angiogenesis under hypoxia were investigated. Via bioinformatic analyses on microarray profiles (GSE1041 and GSE17944), solube fms-related tyrosine kinase 1 (sFLT1, also known as sVEGFR1) and miR-210/miR-424 might be involved in HIF1A function on the angiogenesis under hypoxia in human umbilical vascular endothelium cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs). In the present study, we identified sFLT1 as a downregulated gene in response to hypoxia and HIF1A overexpression in HUVECs and HDMECs. sFLT1 overexpression inhibited the capacity of migration and angiogenesis and significantly reversed the inducible effects of HIF1A on the migration and angiogenesis in both cell lines. miR-210 and miR-424 were upregulated by hypoxia and targeted sFLT1 3'-UTR to negatively modulate its expression. HIF1A modulated sFLT1 expression, VEGF signaling, and the migration and angiogenesis in HUVECs and HDMECs via miR-210/miR-424. Regarding the molecular mechanism, HIF1A bound the promoter region of miR-210 and miR-424 to activate their transcription, while miR-210/miR-424 bound sFLT1 3'-UTR to suppress its expression. In summary, HIF1A/miR-210/miR-424/sFLT1 axis modulates the angiogenesis in HUVECs and HDMECs upon hypoxic condition via VEGF signaling.

Apoptosis induction in human lung and colon cancer cells via impeding VEGF signaling pathways.

There is ample evidence to suggest that vascular endothelial growth factor (VEGF) is a potent mitogen factor in vasculogenesis and angiogenesis and that blockade of VEGF-mediated signals can also prevent tumor growth via enforcing cell apoptosis. In the current study, we assessed the suppressing effect of VGB4, a VEGF antagonist peptide with the binding ability to both VEGF receptor1 and VEGF receptor2, on VEGF-induced proliferation and migration of the human lung adenocarcinoma cell line A549 and the human colon adenocarcinoma cell line HT29 using MTT assay, colony formation assay, and Scratch-wound assay. To evaluate the apoptotic inductive effect of VGB4 on A549 and HT29 cells, apoptosis analysis was carried out by flow cytometry and TUNEL assay. Likewise, p53 and PTEN expression level was examined by immunofluorescence microscopy. In addition, the level of proteins involved in VEGF signaling pathways related to apoptosis was investigated using western blot analysis. Our results indicated that VGB4 markedly inhibited VEGF-induced proliferation and migration, and induced apoptosis of A549 and HT29 cells dose dependently. Encouragingly, significant downregulation of B-cell lymphoma 2 (Bcl2), X-linked inhibitor of apoptosis, Procaspase9, and procaspase3, as well as upregulation of PTEN and P53 tumor suppressors, BCL2 associated X, Cytochrome c, cleaved caspase9, and cleaved caspase3 in VGB4-treated A549 and HT29 cells, further confirmed the profound inductive influence of VGB4 on apoptotic pathways. These findings along with the results from our previous studies show that VGB4 may be considerable for cancer therapy.

Conjugation of VEGFR1/R2-targeting peptide with gold nanoparticles to enhance antiangiogenic and antitumoral activity.

BACKGROUND: Inhibition of tumor angiogenesis through simultaneous targeting of vascular endothelial growth factor receptor (VEGFR)-1 and -2 is highly efficacious. An antagonist peptide of VEGFA/VEGFB, referred to as VGB3, can recognize and neutralize both VEGFR1 and VEGFR2 on the endothelial and tumoral cells, thereby inhibits angiogenesis and tumor growth. However, improved efficacy and extending injection intervals is required for its clinical translation. Given that gold nanoparticles (GNPs) can enhance the efficacy of biotherapeutics, we conjugated VGB3 to GNPs to enhance its efficacy and extends the intervals between treatments without adverse effects. RESULTS: GNP-VGB3 bound to VEGFR1 and VEGFR2 in human umbilical vein endothelial (HUVE) and 4T1 mammary carcinoma cells. GNP-VGB3 induced cell cycle arrest, ROS overproduction and apoptosis and inhibited proliferation and migration of endothelial and tumor cells more effectively than unconjugated VGB3 or GNP. In a murine 4T1 mammary carcinoma tumor model, GNP-VGB3 more strongly than VGB3 and GNP inhibited tumor growth and metastasis, and increased animal survival without causing weight loss. The superior antitumor effects were associated with durable targeting of VEGFR1 and VEGFR2, thereby inhibiting signaling pathways of proliferation, migration, differentiation, epithelial-to-mesenchymal transition, and survival in tumor tissues. MicroCT imaging and inductively coupled plasma mass spectrometry showed that GNP-VGB3 specifically target tumors and exhibit greater accumulation within tumors than the free GNPs. CONCLUSION: Conjugation to GNPs not only improved the efficacy of VGB3 peptide but also extended the intervals between treatments without adverse effects. These results suggest that GNP-VGB3 is a promising candidate for clinical translation.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells.

BACKGROUND: Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. RESULTS: By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. CONCLUSIONS: We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

Knockdown of VEGFB/VEGFR1 Signaling Promotes White Adipose Tissue Browning and Skeletal Muscle Development.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) and vascular endothelial growth factor receptor 1 (VEGFR1) play a vital role in regulating vascular biological function. However, the role of VEGFB and VEGFR1 in regulating fat deposition and skeletal muscle growth remains unclear. Therefore, this study was conducted to investigate the effects of VEGFB and VEGFR1 on fat deposition and skeletal muscle growth in mice. Our results showed that knockdown of VEGFB decreased body weight and iWAT index, stimulated the browning of mice iWAT with increased expression of UCP1, decreased the diameters of adipocytes, and elevated energy expenditure. In contrast, knockdown of VEGFB increased gastrocnemius (GAS) muscle index with increased proliferation of GAS muscle by expression of PCNA and Cyclin D1. Meanwhile, knockdown of endothelial VEGFR1 induced the browning of iWAT with increased expression of UCP1 and decreased diameters of adipocytes. By contrast, knockdown of endothelial VEGFR1 inhibited GAS muscle differentiation with decreased expression of MyoD. In conclusion, these results suggested that the loss of VEGFB/VEGFR1 signaling is associated with enhanced browning of inguinal white adipose tissue and skeletal muscle development. These results provided new insights into the regulation of skeletal muscle growth and regeneration, as well as fat deposition, suggesting the potential application of VEGFB/VEGFR1 as an intervention for the restriction of muscle diseases and obesity and related metabolic disorders.

SNPs in 3'UTR miRNA Target Sequences Associated with Individual Drug Susceptibility.

The complementary interaction of microRNAs (miRNAs) with their binding sites in the 3'untranslated regions (3'UTRs) of target gene mRNAs represses translation, playing a leading role in gene expression control. MiRNA recognition elements (MREs) in the 3'UTRs of genes often contain single nucleotide polymorphisms (SNPs), which can change the binding affinity for target miRNAs leading to dysregulated gene expression. Accumulated data suggest that these SNPs can be associated with various human pathologies (cancer, diabetes, neuropsychiatric disorders, and cardiovascular diseases) by disturbing the interaction of miRNAs with their MREs located in mRNA 3'UTRs. Numerous data show the role of SNPs in 3'UTR MREs in individual drug susceptibility and drug resistance mechanisms. In this review, we brief the data on such SNPs focusing on the most rigorously proven cases. Some SNPs belong to conventional genes from the drug-metabolizing system (in particular, the genes coding for cytochromes P450 (CYP 450), phase II enzymes (SULT1A1 and UGT1A), and ABCB3 transporter and their expression regulators (PXR and GATA4)). Other examples of SNPs are related to the genes involved in DNA repair, RNA editing, and specific drug metabolisms. We discuss the gene-by-gene studies and genome-wide approaches utilized or potentially utilizable to detect the MRE SNPs associated with individual response to drugs.

Quantile-specific heritability of serum growth factor concentrations.

BACKGROUND: "Quantile-dependent expressivity" occurs when the effect size of a genetic variant depends upon whether the phenotype (e.g. growth factor concentration) is high or low relative to its distribution. METHODS: Quantile-regression analysis was applied to family sets from the Framingham Heart Study to determine whether the heritability (h2) of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), angiopoietin-2, and angiopoietin-2 (sTie-2) and VEGFR1 (sFlt-1) receptor concentrations were quantile-specific. RESULTS: Quantile-specific h2 (±SE) increased with increasing percentiles of the age- and sex-adjusted VEGF (Ptrend<10-16), HGF (Ptrend=0.0004), angiopoietin-2 (Ptrend=0.0002), sTie-2 (Ptrend=1.2 × 10-5), and sFlt-1 distributions (Ptrend=0.04). CONCLUSION: Heritabilities of VEGF, HGF, angiopoitein-2, sTie-2 and sFlt-1 concentrations are quantile dependent. This may explain reported interactions of genetic loci (rs10738760, rs9472159, rs833061, rs3025039, rs2280789, rs1570360, rs2010963) with metabolic syndrome, diet, recurrent miscarriage, hepatocellular carcinoma, erysipelas, diabetic retinopathy, and bevacizumab treatment in their effect on VEGF concentrations.

A role for aldehyde dehydrogenase (ALDH) 2 in angiotensin II-mediated decrease in angiogenesis of coronary endothelial cells.

Diabetes-induced coronary endothelial cell (CEC) dysfunction contributes to diabetic heart diseases. Angiotensin II (Ang II), a vasoactive hormone, is upregulated in diabetes, and is reported to increase oxidative stress in CECs. 4-hydroxy-2-nonenal (4HNE), a key lipid peroxidation product, causes cellular dysfunction by forming adducts with proteins. By detoxifying 4HNE, aldehyde dehydrogenase (ALDH) 2 reduces 4HNE mediated proteotoxicity and confers cytoprotection. Thus, we hypothesize that ALDH2 improves Ang II-mediated defective CEC angiogenesis by decreasing 4HNE-mediated cytotoxicity. To test our hypothesis, we treated the cultured mouse CECs (MCECs) with Ang II (0.1, 1 and 10 µM) for 2, 4 and 6 h. Next, we treated MCECs with Alda-1 (10 µM), an ALDH2 activator or disulfiram (2.5 µM)/ALDH2 siRNA (1.25 nM), the ALDH2 inhibitors, or blockers of angiotensin II type-1 and 2 receptors i.e. Losartan and PD0123319 respectively before challenging MCECs with 10 µM Ang II. We found that 10 µM Ang II decreased tube formation in MCECs with in vitro angiogenesis assay (P < .0005 vs control). 10 µM Ang II downregulated the levels of vascular endothelial growth factor receptor 1 (VEGFR1) (p < .005 for mRNA and P < .05 for protein) and VEGFR2 (p < .05 for mRNA and P < .005 for protein) as well as upregulated the levels of angiotensin II type-2 receptor (AT2R) (p < .05 for mRNA and P < .005 for protein) and 4HNE-adducts (P < .05 for protein) in cultured MCECs, compared to controls. ALDH2 inhibition with disulfiram/ALDH2 siRNA exacerbated 10 µM Ang II-induced decrease in coronary angiogenesis (P < .005) by decreasing the levels of VEGFR1 (P < .005 for mRNA and P < .05 for protein) and VEGFR2 (P < .05 for both mRNA and protein) and increasing the levels of AT2R (P < .05 for both mRNA and protein) and 4HNE-adducts (P < .05 for protein) relative to Ang II alone. AT2R inhibition per se improved angiogenesis in MCECs. Additionally, enhancing ALDH2 activity with Alda 1 rescued Ang II-induced decrease in angiogenesis by increasing the levels of VEGFR1, VEGFR2 and decreasing the levels of AT2R. In summary, ALDH2 can be an important target in reducing 4HNE-induced proteotoxicity and improving angiogenesis in MCECs. Finally, we conclude ALDH2 activation can be a therapeutic strategy to improve coronary angiogenesis to ameliorate cardiometabolic diseases.

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.

Role of VEGFR-1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF-A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR-1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour-associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro-angiogenic pathways, we have investigated VEGFR-1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF-A and express higher VEGFR-1 levels compared with their BRAFi-sensitive counterparts. Transient VEGFR-1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR-1 expression, by stable gene transfection in receptor-negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR-1 are more invasive than VEGFR-1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF-A and PlGF. These data suggest that VEGFR-1 up-regulation might contribute to melanoma progression and spreading after acquisition of a drug-resistant phenotype. Thus, VEGFR-1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR-1 positive tumours with acquired resistance to vemurafenib.

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

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