Vascular Endothelial Growth Factor Stimulation Research Articles

Inflammation mediated angiogenesis in chronic lymphocytic leukemia.

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

The effect of VEGF stimulation in diabetic foot ulcers: A systematic review.

Diabetic foot ulcers are a common and severe complication of diabetes mellitus, and a risk factor for amputation. Because of the vessel insufficiency in diabetic foot ulcers (DFU), vascular endothelial growth factor (VEGF) that simulates angiogenesis is of interest to promote wound healing. This systematic review evaluates the last 16 years of in-vivo studies with VEGF stimulation as a treatment for DFU, developed based on the last published systematic article. A total of 961 articles were identified through databases in two phases. 947 articles were excluded by exclusion criteria, and four articles met our inclusion criteria and were included. The effects of VEGF on wound healing were analysed in all four studies. In three studies, the VEGF-treated wounds showed statistically faster healing than those not treated with VEGF. In one study, the VEGF-treated wounds revealed a positive trend toward faster healing. Furthermore, all four studies were in favor of using VEGF, but concluded that further research is needed. These studies showed a positive trend towards faster healing and was safe when using VEGF topically on humans. Furthermore, viral particles of VEGF seem to have a systematic effect when a dose exceeding 5.0 × 109 vp pr wound. Future research in using VEGF on DFU should focus on VEGF's relevant dosage, release rate, and specific mechanism. This review inspires further research, and a consistent study design is prerequisite such that results are more homogenic and comparable. Much effort is needed to translate the results into our clinical practice.

A novel small molecule, CU05-1189, targeting the pleckstrin homology domain of PDK1 suppresses VEGF-mediated angiogenesis and tumor growth by blocking the Akt signaling pathway.

Inhibition of angiogenesis is considered a promising therapeutic approach for cancer treatment. Our previous genetic research showed that the use of a cell-penetrating peptide to inhibit the pleckstrin homology (PH) domain of 3-phosphoinositide-dependent kinase 1 (PDK1) was a viable approach to suppress pathological angiogenesis. Herein, we synthesized and characterized a novel small molecule, CU05-1189, based on our prior study and present evidence for the first time that this compound possesses antiangiogenic properties both in vitro and in vivo. The computational analysis showed that CU05-1189 can interact with the PH domain of PDK1, and it significantly inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells without apparent toxicity. Western blot analysis revealed that the Akt signaling pathway was specifically inhibited by CU05-1189 upon VEGF stimulation, without affecting other VEGF receptor 2 downstream molecules or cytosolic substrates of PDK1, by preventing translocation of PDK1 to the plasma membrane. We also found that CU05-1189 suppressed VEGF-mediated vascular network formation in a Matrigel plug assay. More importantly, CU05-1189 had a good pharmacokinetic profile with a bioavailability of 68%. These results led to the oral administration of CU05-1189, which resulted in reduced tumor microvessel density and growth in a xenograft mouse model. Taken together, our data suggest that CU05-1189 may have great potential and be a promising lead as a novel antiangiogenic agent for cancer treatment.

Ebola virus disrupts the inner blood-retinal barrier by induction of vascular endothelial growth factor in pericytes.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans with high mortality. In Ebola virus disease (EVD) survivors, EBOV persistence in the eyes may break through the inner blood-retinal barrier (iBRB), leading to ocular complications and EVD recurrence. However, the mechanism by which EBOV affects the iBRB remains unclear. Here, we used the in vitro iBRB model to simulate EBOV in retinal tissue and found that Ebola virus-like particles (EBO-VLPs) could disrupt the iBRB. Cytokine screening revealed that EBO-VLPs stimulate pericytes to secrete vascular endothelial growth factor (VEGF) to cause iBRB breakdown. VEGF downregulates claudin-1 to disrupt the iBRB. Ebola glycoprotein is crucial for VEGF stimulation and iBRB breakdown. Furthermore, EBO-VLPs caused iBRB breakdown by stimulating VEGF in rats. This study provides a mechanistic insight into that EBOV disrupts the iBRB, which will assist in developing new strategies to treat EBOV persistence in EVD survivors.

Platelet-derived growth factor (PDGF) cross-signaling via non-corresponding receptors indicates bypassed signaling in colorectal cancer.

Platelet-derived growth factor (PDGF) signaling, besides other growth factor-mediated signaling pathways like vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), seems to play a crucial role in tumor development and progression. We have recently provided evidence for upregulation of PDGF expression in UICC stage I–IV primary colorectal cancer (CRC) and demonstrated PDGF-mediated induction of PI3K/Akt/mTOR signaling in CRC cell lines. The present study sought to follow up on our previous findings and explore the alternative receptor cross-binding potential of PDGF in CRC. Our analysis of primary human colon tumor samples demonstrated upregulation of the PDGFRβ, VEGFR1, and VEGFR2 genes in UICC stage I-III tumors. Immunohistological analysis revealed co-expression of PDGF and its putative cross-binding partners, VEGFR2 and EGFR. We then analyzed several CRC cell lines for PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 protein expression and found these receptors to be variably expressed amongst the investigated cell lines. Interestingly, whereas Caco-2 and SW480 cells showed expression of all analyzed receptors, HT29 cells expressed only VEGFR1 and VEGFR2. However, stimulation of HT29 cells with PDGF resulted in upregulation of VEGFR1 and VEGFR2 expression despite the absence of PDGFR expression and mimicked the effect of VEGF stimulation. Moreover, PDGF recovered HT29 cell proliferation under simultaneous treatment with a VEGFR or EGFR inhibitor. Our results provide some of the first evidence for PDGF cross-signaling through alternative receptors in colorectal cancer and support anti-PDGF therapy as a combination strategy alongside VEGF and EGF targeting even in tumors lacking PDGFR expression.

Effect of platelet-rich plasma combined with tranexamic acid in the treatment of melasma and its effect on the serum levels of vascular endothelial growth factor, endothelin-1 and melatonin.

To investigate the effect of platelet-rich plasma (PRP) combined with tranexamic acid (TXA) in the treatment of melasma and its effect on the serum levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and melanin stimulating hormone (MSH). We retrospectively analyzed clinical data of 80 patients with melasma treated in our hospital from January 2020 to June 2021. Patients (n=38) in the control group received simple oral TXA treatment. Patients (n=42) in the study group received PRP combined with oral TXA treatment. We assessed the treatment effects on the serum biochemical index levels, the adverse reactions, and the recurrence rates in the two groups. The total efficacy of the study group (90.48%) was higher than that of the control group (73.68%) (p<0.05). After the treatment, the levels of serum VEGF increased and the levels of ET-1 and MSH decreased in both groups, but the changes in the study group were more pronounced than those in the control group (p<0.05). We found similar incidences of the adverse reaction in the study group (7.14%) and the control group (5.26%; p>0.05). The disease recurrence rates between the two groups three months after the treatment were similar (p>0.05). However, the disease recurrence rate in the study group (4.76%) was lower than that in the control group (21.05%) 6 months after treatment (p<0.05). PRP combined with oral TXA can improve the treatment effect of TXA alone in the treatment of melasma, maintaining normal levels of VEGF, ET-1 and MSH, reducing disease recurrences.

PLCβ2 Promotes VEGF-Induced Vascular Permeability.

Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) β2. Global knock-out of PLCβ2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCβ2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCβ2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCβ2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. The results implicate PLCβ2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCβ2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Abstract 190: Deletion Of Shp-1 Prevents Diabetes-induced Endothelial Cell Senescence And Restores Blood Flow Reperfusion Following Ischemia

Introduction: Ischemia due to narrowing of the femoral artery and distal vessels is also a major cause of peripheral arterial disease and morbidity affecting patients with diabetes. Diabetes-induced premature senescence of endothelial cells has been shown as a potential mechanism of poor angiogenic response to ischemia. Our laboratory has previously shown that hyperglycemia reduced vascular endothelial growth factor (VEGF) activity in ischemic muscle of diabetic mice, which was associated with increased SHP-1 expression, a protein tyrosine phosphatase. The objective of this study is to evaluate the impact of SHP-1 deletion on endothelial cell function and senescence both in vitro and in vivo . Methods: Non-diabetic (NDM) and 3 months diabetic (DM) mice with deletion of SHP-1 specifically in endothelial cells (EC) were used. Ligation of the femoral artery was performed, and blood flow reperfusion was measured by laser Doppler for 4 weeks. Primary EC were exposed to normal (5.6 mM; NG) or high glucose concentrations (25 mM; HG) for 48 h, in normoxia (20% oxygen) or hypoxia (1%) for the last 16h in presence of VEGF. Results: Blood flow reperfusion and limb function (voluntary running wheel) were reduced by 50% and 91%, respectively in DM mice as compared to NDM mice. Specific EC deletion of SHP-1 restored blood flow reperfusion by 55%, limb function by 37% and capillary density in DM mice. Moreover, ablation of SHP-1 only in EC prevented diabetes-induced p21 expression and reduction of Nrf-2. In cultured EC , overexpression of dominant negative of SHP-1 prevented HG-induced inhibition of proliferation, migration, and VEGFR2/Akt phosphorylation following VEGF stimulation. In addition, the expression of senescence markers (increased p21 and beta-galactosidase; reduced Nrf-2) in EC exposed to HG levels were reversed by overexpression of SHP-1 dominant negative. Conclusion: Ablation of SHP-1 expression in EC resulted in the prevention of hyperglycemia-induced EC senescence, reduction of VEGF actions, poor collateral vessel formation and blood flow reperfusion.

Influence of vascular endothelial growth factor on neutrophil activation in asthmatics.

IntroductionAsthma is a complex syndrome associated with heterogeneity in the type of inflammation that modulates airway hyperresponsiveness and remodelling. Airway inflammation with neutrophils, cytokines like vascular endothelial growth factor (VEGF) and various gene polymorphisms might be relevant for asthma pathogenesis.AimTo investigate the expression of CD69 and CD11b markers on peripheral neutrophils after VEGF in vitro stimulation in asthmatics. Furthermore, the possible influence of a genetic factor (del/ins genotype at -2549 -2567 position in the VEGF-promoter gene) was taken into account.Material and methods122 subjects (82 asthmatics and 40 controls) participated in our study. CD69 and CD11b presence on peripheral blood neutrophils was detected using the flow cytometric method without exogenous stimulation (negative control), after N-formyl-methionine-leucyl-phenylalanine stimulation (fMLP – positive control) and after VEGF stimulation. Genotyping for the VEGF-promoter region was performed by polymerase chain reaction (PCR).ResultsPeripheral neutrophil stimulation with VEGF enhances the activity of these cells and induces CD69 and CD11b expression in a dose-dependent manner compared with unstimulated neutrophils (p > 0.05). CD69 and CD11b markers were slightly presented (p = 0.05 and p = 0.07, respectively) on neutrophils stimulated with fMLP in asthmatics with the ins genotype variant in the VEGF-promoter region.ConclusionsOur results demonstrate that VEGF might insignificantly activate neutrophils in asthmatics. In addition, the modulated expression of CD69 and CD11b on peripheral neutrophils is not related to potential contribution of the VEGF gene polymorphism.

Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment.

T-type Ca2+ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type Ca2+ channel isoforms, Cav3.1 (encoded by Cacna1g), Cav3.2 (encoded by Cacna1h), and Cav3.3 (encoded by Cacna1i), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type Ca2+ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type Ca2+ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity.

S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis.

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. Vascular endothelial growth factor (VEGF) stimulation of endothelial progenitor cells (EPCs) facilitates angiogenesis and the progression of RA. Phosphorylation of sphingosine kinase 1 (SphK1) produces sphingosine-1-phosphate (S1P), which increases inflammatory cytokine production, although the role of S1P in RA angiogenesis is unclear. In this study, we evaluated the impact of S1P treatment on VEGF-dependent angiogenesis in osteoblast-like cells (MG-63 cells) and the significance of SphK1 short hairpin RNA (shRNA) on S1P production in an in vivo model. We found significantly higher levels of S1P and VEGF expression in synovial fluid from RA patients compared with those with osteoarthritis by ELISA analysis. Treating MG-63 cells with S1P increased VEGF production, while focal adhesion kinase (FAK) and Src siRNAs and inhibitors decreased VEGF production in S1P-treated MG-63 cells. Conditioned medium from S1P-treated osteoblasts significantly increased EPC tube formation and migration by inhibiting miR-16-5p synthesis via proto-oncogene tyrosine-protein kinase src (c-Src) and FAK signaling in chick chorioallantoic membrane (CAM) and Matrigel plug assays. Infection with SphK1 shRNA reduced angiogenesis, articular swelling and cartilage erosion in the ankle joints of mice with collagen-induced arthritis (CIA). S1P appears to have therapeutic potential in RA treatment.

Effects of serum starvation and vascular endothelial growth factor stimulation on the expression of Notch signalling pathway components.

Brain arteriovenous malformation (BAVM) is an abnormality in the cerebral vascular system. Although the upregulation of the Notch signalling pathway is a deterministic factor in BAVM, the mechanism by which this pathway is upregulated in patients with BAVM is uncertain. The effects of serum starvation and vascular endothelial growth factor (VEGF) stimulation on the Notch signalling pathway in brain microvascular endothelial cells (MECs) and mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells were investigated in this study. The duration of serum starvation and VEGF concentration were changed, cell viability was measured, and reasonable time and concentration gradients were selected for subsequent studies. Protein and mRNA expression levels of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells were detected using western blotting and real-time PCR, respectively. Expression levels of the Notch1, Notch4, Jagged1, delta-like ligand 4 (Dll4) and Hes1 proteins and mRNAs were upregulated by lower VEGF concentrations and shorter-term serum starvation but inhibited by higher VEGF concentrations and longer-term serum starvation. This study revealed effects of changes in the duration of serum starvation and VEGF concentration on the expression of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells, potentially contributing to BAVM formation.

A Bilayered, Electrospun Poly(Glycerol-Sebacate)/Polyurethane-Polyurethane Scaffold for Engineering of Endothelial Basement Membrane.

In the cardiovascular system, heart valves and vessels are subjected to continuous cyclic mechanical loadings due to the pulsatile nature of blood flow. Hence, in leveraging tissue engineering (TE) strategies to regenerate such a system, the candidate scaffold should not only be biocompatible with the desired biodegradation rate, but it should also be mechanically competent to provide a supportive structure for facilitating stem cells retention, growth, and differentiation. To this end, herein, we introduced a novel scaffold composed of poly(glycerol-sebacate) (PGS) and polyurethane (PU), which comprises of two layers: an electrospun pure PU layer beneath another electrospun PGS/PU layer with a different ratio of PGS to PU (3:2, 1:1, 2:3 Wt:Wt). The electrospun PGS/PU-PU scaffold was mechanically competent and showed intended hydrophilicity and a good biodegradation rate. Moreover, the PGS/PU-PU scaffold indicated cell viability and proliferation within ten days of in vitro cell culture and upon 7 day vascular endothelial growth factor (VEGF) stimulation, supported endothelial differentiation of mesenchymal stem cells by significant overexpression of platelet-endothelial cell adhesion molecule-1, von Willebrand factor, and VEGF receptor 2. The results of this study could be implemented in cardiovascular TE strategies when regeneration of blood vessel or heart valve is desired.

Protein Phosphatase 2A Mediates YAP Activation in Endothelial Cells Upon VEGF Stimulation and Matrix Stiffness.

Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis.

Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.

In the lungs, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological responses remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lungs and thus assumed to be the same as the one acting on the alveolar epithelium. Existing in vitro models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced in vitro vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature in vitro. We studied the effects of the cyclic strain on a perfusable 3D vasculature, made of either human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The in vitro 3D vessels underwent both in vivo-like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with the 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however, we observed several phenomena in the 3D model that the 2D model was unable to predict. Angiogenic sprouting induced by VEGF decreased significantly in the presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast to our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer, which truly recapitulates the in vivo situation.

Inhibitory Effects of Combined Bone Morphogenetic Protein 2, Vascular Endothelial Growth Factor, and Basic Fibroblast Growth Factor on Osteoclast Differentiation and Activity.

Bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factors (bFGF) are important regulators of bone development and bone remodeling involving the coordination of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The synergistic promotions of these growth factors on osteogenesis in the appropriate combination have been confirmed by a lot of studies, but the effect of this combined application on osteoclastogenesis still remains ambiguous. On the basis of comparing the osteoclastic potentials under stimulation of BMP-2, VEGF, or bFGF alone, this study focused on their combined effects on the differentiation and activity of osteoclasts. Our results showed that osteoclastogenesis was enhanced to some extent under the stimulation of BMP-2, VEGF, or bFGF alone, and the potential of these three growth factors to stimulate osteoclastogenesis was VEGF > BMP-2 > bFGF. However, the treatment with the combination of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL), the most suitable dose combination for osteogenesis optimized in our previous study, weakened osteoclast differentiation confirmed by smaller tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, lower TRAP activity, and lower expression of dendritic cell-specific transmembrane protein, an important molecule regulating osteoclast fusion. Moreover, BMP-2, VEGF, and bFGF in combination also moderately inhibited the bone-resorbing activity of mature osteoclasts by suppressing the expression of osteoclast-specific genes cathepsin K, and matrix metalloproteinase-9. The underlying molecular mechanisms involved the suppression of the receptor activator of nuclear factor-κB ligand-induced c-Fos levels and the activation of nuclear factor of activated T cells c1, two major transcription factors in osteoclast differentiation. Taken together, our study showed that the combination of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL) promoted osteoblastogenesis but inhibited osteoclastogenesis. Thus, the simultaneous use of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL) in an appropriate combination might improve efficacious bone regeneration in a clinical setting. Impact statement Few studies have addressed the combined effects of multiple growth factors on osteoclasts. This study demonstrated that the simultaneous use of bone morphogenetic protein 2 (BMP-2; 50 ng/mL), vascular endothelial growth factor (VEGF; 1 ng/mL), and basic fibroblast growth factors (bFGF; 10 ng/mL), the most suitable dose combination for osteogenesis optimized in our previous study, showed inhibitory effects on the differentiation and activity of osteoclasts. Our results suggest that the growth factor signaling pathways in osteoclasts may interact with each other. Furthermore, this study could provide new insights into the optimal application of BMP-2, VEGF, and bFGF for bone repair and regeneration.

Abstract A24: CD146-Rictor interaction reveals a pathway linking mTORC2 activation with extracellular stimuli

Abstract In all eukaryotes, the mechanistic target of rapamycin (mTOR) signaling emerges as a master regulator of homeostasis, which integrates environmental inputs to regulate many fundamental cellular processes such as cell growth and metabolism. The mTOR signaling functions through two structurally and functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which correspond to two major branches of signal output. However, it remains enigmatic how extracellular stimuli regulates mTOR signaling, particularly for mTORC2. Here, we demonstrate that in human umbilical vein endothelial cells (HUVEC), vascular endothelial growth factor (VEGF) promotes the activation of mTORC2 signaling, exhibited as phosphorylation of Akt /PKCα/SGK1. Cell adhesion molecule CD146, as VEGFR2 co-receptor, plays the pivotal role in mediating mTORC2 activation in response to VEGF. Deletion of CD146 resulted in dramatic reduction in mTORC2 activity, without affecting other PI3K/Akt/mTOR signaling pathways, such as mTORC1 and PI3K/PDK1, indicating that CD146 specifically regulates mTORC2 activity. By means of co-immunoprecipitation (co-IP) assay, CD146 is bestowed to interact with Rictor via its juxta-membrane KKGK motif upon VEGF stimulation, CD146-Rictor (mTORC2) interaction not only stabilizes Rictor from ubiquitin-proteasome-mediated degradation but also activates mTORC2 signaling, thus promoting VEGF-induced endothelial cell proliferation and survival. Collectively, our findings have identified a previously unanticipated mechanism by which mTORC2 signaling pathway is directly under the control of extracellular stimuli, linking environmental cues with mTORC2 activity, and implicate that targeting CD146-mTORC2 axis might be a novel strategy for diseases driven by uncontrolled cell proliferation. Citation Format: Wenyi Xu, Yongting Luo, Wei Cui. CD146-Rictor interaction reveals a pathway linking mTORC2 activation with extracellular stimuli [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A24.

Characterization of tryptophan‐containing dipeptides for anti‐angiogenic effects

In the pathogenesis of several diseases, neo-angiogenesis is increased (e.g. tumour growth). The peptide L-glutamyl-L-tryptophan (EW/IM862) has been claimed to exhibit inhibitory effects on tumour growth in vivo. However, the potential role of natural peptides with respect to anti-angiogenic properties is unsettled. The current study explores anti-angiogenic effects of the dipeptides WL, EW, IW and WE. Using a bottom-up strategy, we first evaluated the effects of the peptides on VEGFR-2 signalling and quantified their effects in different angiogenesis assays. WL consistently had the strongest effects on phosphorylation of VEGFR-2 and downstream signalling. Therefore, this peptide was chosen in comparison with EW to further assess anti-angiogenic properties. However, sprout formation in three-dimensional (3D) fibrin gel bead assay was significantly inhibited by EW only. Furthermore, vessel sprouting in the mouse aortic ring assay was decreased by the presence of WL and EW compared to control. Results from a chorioallantoic membrane assay showed that under vascular endothelial growth factor (VEGF) stimulation WL and EW decreased the number of blood vessels versus control. These results were in line with those obtained in a matrigel plug assay. The VEGF-induced increase in the haemoglobin content was nearly abolished when treatment was combined with either WL or EW application. In the murine model of oxygen-induced retinopathy, WL exhibited a small albeit significant anti-angiogenic effect. Comprehensive screening of WL suggests an anti-angiogenic effect, demonstrated in in vitro, ex vivo and in vivo models. Thus, WL is a dipeptide with potential anti-angiogenic effects and is worthy for further exploration.

Targeted delivery of mitomycin C-loaded and LDL-conjugated mesoporous silica nanoparticles for inhibiting the proliferation of pterygium subconjunctival fibroblasts

Pterygium is a degenerative disease that characterized by excessive fibrovascular proliferation. To reduce the recurrence rate, surgery is the main strategy, in combination with adjacent procedures or adjunctive therapy. One of the most common adjunctive agents, mitomycin C (MMC), is known as an alkylating agent that inhibits fibroblast proliferation but is limitedly applied in pterygium due to various complications. A previous study demonstrated that activated pterygium subconjunctival fibroblasts overexpressed low-density lipoprotein (LDL) receptors. In this study, we designed and synthesized MMC-loaded mesoporous silica nanoparticles conjugated with LDL (MMC@MSNs-LDL) to deliver MMC into activated pterygium fibroblasts in a targeted manner. The MMC loading efficiency was approximately 6%. The cell viability test (CCK-8 assay) revealed no cytotoxicity for the empty carrier MSNs at a concentration of ≤1 mg/ml after administration for 48 h in subconjunctival fibroblasts. Primary pterygium and normal human subconjunctival fibroblasts with or without stimulation by vascular endothelial growth factor (VEGF) were treated as follows: 1) 10 μg/ml MMC@MSNs-LDL for 24 h (MMC concentration: 0.6 μg/ml); 2) 0.2 mg/ml MMC for 5 min then cultured for 24 h after MMC removal; and 3) normal culture without any drug treatment. At 24 h, the anti-proliferative effect of MMC@MSNs-LDL in activated pterygium fibroblasts was similar to that of MMC (cell viability: 46.2 ± 5.5% vs 40.5 ± 1.1%, respectively, P = 0.349). Furthermore, the cytotoxicity of MMC@MSNs-LDL to normal fibroblasts with or without VEGF stimulation was significantly lower than that of traditional MMC (cell viability: 75.6 ± 4.4% vs 36.0 ± 1.5%, respectively, P < 0.001; 84.7 ± 5.5% vs 35.7 ± 1.3%, P < 0.001). The binding of fluorescently labeled MMC@MSNs-LDL in fibroblasts was assessed using confocal fluorescence microscopy. The uptake of targeted nanoparticles in fibroblasts was time dependent and saturated at 6 h. VEGF-activated pterygium fibroblasts showed more uptake of MMC@MSNs-LDL than normal fibroblasts with or without VEGF activation (both P < 0.001). Our data strongly suggest that MMC@MSNs-LDL had an effective antiproliferative role in activated pterygium fibroblasts, with reduced toxicity to normal fibroblasts compared to traditional application of MMC. LDL-mediated drug delivery might have great potential in the management of pterygium recurrence.

Intravenous Administration is the Best Route of Mesenchymal Stem Cells Migration in Improving Liver Function Enzyme of Acute Liver Failure

Background: Mesenchymal stem cells (MSCs) migrate and transmigrate to acute liver failure (ALF) area due to vascular endothelial growth factor (VEGF) stimulation as an attractant molecule then actively giving the paracrine signaling and or differentiating into primary hepatocytes, however the best route of MSCs transplanted to liver injury area remains unclear. &amp;nbsp; Aim: In this study we compare intravenous (IV) and intraperitoneal (IP) route of MSCs administration by analyzing serum glutamic pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT) and bilirubin level as improvement markers of liver function and VEGF as attractant-proliferation molecule on days 2 and 5. &amp;nbsp; Materials and methods: Eighteen male Sprague-Dawley rats weighting 200 g were used in this study. They were divided in three study groups: vehicle control, IP and IV groups. The IV group was treated by MSCs at dose 1&amp;times;106 by lateral tail vein injection and IP group received 1&amp;times;106 MSCs via IP injection. The level of SGPT, SGOT and bilirubin were measured by an automatic analyzer, the VEGF level using enzyme-linked immunosorbent assay (ELISA), while the CD73 expression was evaluated using immunohistochemistry. &amp;nbsp; Results: This study showed that IV injection of MSCs was more efficient for increasing liver function than IP treatment group that confirmed by the observed significant decrease in SGPT, SGOT and bilirubin level on days 2 and 5 (p&lt;0.001). This effect was most likely mediated by the significant increase of VEGF level (p&lt;0.05) on days 2 and 5. &amp;nbsp; Conclusion: Our result conclude that an IV administration of MSCs was more efficacious than the IP administration for liver injury &amp;nbsp;regeneration.&amp;nbsp;