Vascular Endothelial Cell Growth factor-A Research Articles

XSSJS inhibits hepatic fibrosis by promoting the miR-29b-3p/VEGFA axis in vitro and in vivo.

Hepatic pathological angiogenesis (HPA) is the key event of hepatic fibrosis (HF). Xueshisanjia powder (XSSJS), a Chinese herbal compound, is beneficial for alleviating pathological angiogenesis of hepatic tissue. The present study attempts to reveal the effect and mechanism of XSSJS via regulating miR-29b-3p/VEGFA axis against pathological angiogenesis in HF. In in vitro model, human embryonic kidney 293T cells were transfected with miR-29b-3p mimics, whereby the expression of miR-29b-3p was tested by real-time quantitative polymerase chain reaction (RT-qPCR), ensued by Luciferase assay determining the relationship between miR-29b-3p and vascular endothelial cell growth factor A (VEGFA). In addition, miR-29b-3p mimic transfected into the activated hepatic stellate cell T6 (HSC-T6). The Cell-Counting-Kit 8 (CCK8) and 5-Bromodeoxyuridine (BrdU) staining were first utilized to detect the antiproliferative efficiency of XSSJS following the XSSJS compound serum intervention, and then used to observe the expression of transforming growth factor-β (TGF-β), VEGFA, platelet-derived growth factor (PDGF) via RT-PCR, Western blot (WB), and Immunofluorescence (IF) methods. During the in vivo model, XSSJS with boil-free granules were fed to Wistar rats with liver fibrosis caused by intraperitoneal injection of pig serum followed by the transfection of miR-29b-3p adeno-associated virus (AAV). Hematoxylin–Eosin (HE) staining was used for histopathology assessment. The expression of miR-29b-3p, VEGFA, PDGF, TGF-β have been investigated in liver tissue using RT-PCR, WB, IF. The results verified that XSSJS could up-regulate miR-29b-3p and suppress the expression of VEGFA, PDGA, and TGF-β. In mechanism, miR-29b-3p primarily targeted the 3′UTR of VEGFA. In conclusion, XSSJS could modulate miR-29b-3p/VEGFA axis to inhibit the pathological angiogenesis of HF.

Vascular Endothelial Cells Produce Coagulation Factors That Control Their Growth via Joint Protease-Activated Receptor and C5a Receptor 1 (CD88) Signaling

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.

Rapamycin Eyedrops Increased CD4+Foxp3+ Cells and Prevented Goblet Cell Loss in the Aged Ocular Surface.

Dry eye disease (DED), one of the most prevalent conditions among the elderly, is a chronic inflammatory disorder that disrupts tear film stability and causes ocular surface damage. Aged C57BL/6J mice spontaneously develop DED. Rapamycin is a potent immunosuppressant that prolongs the lifespan of several species. Here, we compared the effects of daily instillation of eyedrops containing rapamycin or empty micelles for three months on the aged mice. Tear cytokine/chemokine profile showed a pronounced increase in vascular endothelial cell growth factor-A (VEGF-A) and a trend towards decreased concentration of Interferon gamma (IFN)-γ in rapamycin-treated groups. A significant decrease in inflammatory markers in the lacrimal gland was also evident (IFN-γ, IL-12, CIITA and Ctss); this was accompanied by slightly diminished Unc-51 Like Autophagy Activating Kinase 1 (ULK1) transcripts. In the lacrimal gland and draining lymph nodes, we also observed a significant increase in the CD45+CD4+Foxp3+ cells in the rapamycin-treated mice. More importantly, rapamycin eyedrops increased conjunctival goblet cell density and area compared to the empty micelles. Taken together, evidence from these studies indicates that topical rapamycin has therapeutic efficacy for age-associated ocular surface inflammation and goblet cell loss and opens the venue for new investigations on its role in the aging process of the eye.

Amelioration of testosterone-induced benign prostatic hyperplasia using febuxostat in rats: The role of VEGF/TGFβ and iNOS/COX-2

Benign prostatic hyperplasia (BPH) is a common male disorder. Febuxostat is a non-purine, selective inhibitor of xanthine oxidase (XO), which has a strong antioxidant capacity and pleiotropic pharmacological properties. This study's objective was to explore the potential ameliorative effects of febuxostat against testosterone-induced BPH in rats. Febuxostat (10 mg/kg/day, per os [p.o.]) prevented increased prostate index levels, serum levels of prostate-specific antigen (PSA), and testosterone levels compared to animals treated with testosterone alone, when administered for 28 days. Histological examination indicated that febuxostat dramatically ameliorated pathological changes in the prostate architecture compared to the testosterone group. Similarly, febuxostat markedly improved testosterone-induced oxidative stress by inhibiting the increase in lipid peroxide and nitrite content, and by reducing the level of depletion of reduced glutathione (GSH) and superoxide dismutase (SOD) activity, which significantly reduced the prostate content of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Furthermore, febuxostat significantly reduced the prostatic content, both in terms of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) messenger ribonucleic acid (mRNA) levels, and of protein levels. Moreover, compared to the testosterone group, febuxostat's beneficial effects prevented the increase in growth factors, comprising vascular endothelial cell growth factor A (VEGF-A) and transforming growth factor beta (TGF-β) protein levels. Its ameliorating effects were equal to those of finasteride, which is the most widely used remedy for BPH. In conclusion, this study provides novel evidence that febuxostat experimentally attenuates testosterone-induced BPH in rats, at least in part by inhibiting iNOS/COX-2 and VEGF/TGF-β pathways.

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA.

Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5' terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA

AbstractCurrently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light‐controlled crRNA by coupling vitamin E and a photolabile linker at the 5′ terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E‐caged crRNA was successfully activated to achieve light‐induced genome editing of vascular endothelial cell‐growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9‐mediated gene editing.

Ranibizumab Alters Levels of Intraocular Soluble Cytokine Receptors in Patients with Diabetic Macular Edema

ABSTRACTPurpose: Ranibizumab, an anti-VEGF-A (vascular endothelial cell growth factor-A) monoclonal antibody fragment, is a well-established treatment for diabetic patients with macular edema. However, very little is known about the effect of ranibizumab on intraocular regulation of pro – and anti-inflammatory signaling pathways and their regulation of VEGF family members, which was the aim of this study.Materials and Methods: Diabetic patients (n = 10) aged ≥18 years with central diabetic macular edema, BCVA >24 and <78, and central macular thickness (CMT) greater than 250 μm were enrolled in this study. Following a full eye exam, imaging, and an aqueous tap, patients received ranibizumab (0.3 mg/0.05 mL) injections at day one and weeks four and eight. At week 12, a full eye exam, imaging, and a second aqueous tap was obtained prior to the last injection of ranibizumab. Pre – and post-treatment aqueous humor samples were then analyzed using Milliplex MAP magnetic bead assays.Results: As expected, ranibizumab lowered levels of VEGF-A, decreased CMT, and improved VA (visual acuity). In addition, it significantly lowered aqueous levels of IL-10, IFNγ, sIL-1R1, sIL-1R2, sRAGE, and VEGF-D. Changes in levels of VEGF-A and VEGF-C strongly correlated with changes in soluble receptors, sgp130 and sIL-6R, associated with IL-6 signaling pathways. In contrast, changes in VEGF-D correlated with sIL-1R1 and sIL-1R2, soluble receptors participating in IL-1 signaling. Changes in CMT and VA did not correlate with changes in levels of VEGF family members. However, post-treatment values of CMT correlated with post-treatment levels of VEGF-C. Post-treatment VA values correlated with a wide variety of potential biomarkers linked to inflammation.Conclusions: Ranibizumab treatment had strong effects on regulating levels of soluble receptors closely linked to IL-1 and IL-6 signaling pathways. Therefore, a complete understanding of the actions of ranibizumab will further the development of additional therapies to support treatment of diabetic macular edema.

Circular RNA circSCAF11 Accelerates the Glioma Tumorigenesis through the miR-421/SP1/VEGFA Axis

Circular RNAs (circRNAs) are a novel category of non-coding RNAs, and they have been identified to participate in glioma tumorigenesis. Here we investigated the functions of circRNA circSCAF11 in glioma genesis, and we unveiled its molecular mechanism in the pathophysiological process. Expression levels of circSCAF11, miR-421, and SP1 mRNA were measured using RT-PCR. Proteins were measured using western blotting. The tumor phenotypes of glioma cells were detected using flow cytometry and Cell Counting Kit-8 (CCK-8), transwell, and xenograft mouse assays. The combination within circSCAF11, miR-421, and SP1 was validated using luciferase reporter assay or RNA pull-down assay. The binding of transcription factor SP1 with vascular endothelial cell growth factor A (VEGFA) promoter was inspected using chromatin immunoprecipitation (ChIP). circSCAF11 expression was found to be significantly upregulated in the glioma tissue specimens and cell lines. The ectopic overexpression of circSCAF11 was closely correlated with the poor clinical outcome of glioma patients. Functionally, knockdown of circSCAF11 inhibited the proliferation, invasion, and tumor growth and induced the G0/G1 phase arrest. Mechanically, circSCAF11 positively regulated the SP1 expression through sponging miR-421. Moreover, transcription factor SP1 activated the transcription of VEGFA, constructing the circSCAF11/miR-421/SP1/VEGFA axis in the glioma genesis. The findings in this research illustrate that circSCAF11 accelerates glioma tumorigenesis through the miR-421/SP1/VEGFA axis, providing a potential target for circRNA and glioma treatment.

Angioplasty Using 4-Hexylresorcinol-Incorporated Silk Vascular Patch in Rat Carotid Defect Model

The aim of this study was to evaluate and compare the efficacy of 4-hexylresorcinol (4-HR)-incorporated silk as a vascular patch scaffold to that of the commercial polytetrafluoroethylene (PTFE) vascular patch (GORE® ACUSEAL). The expression of the vascular endothelial cell growth factor-A (VEGF-A) after application of 4-HR was studied in RAW264.7 and HUVEC cells. In the animal study, a carotid artery defect was modeled in Sprague Dawley rats (n = 30). The defect was directly closed in the control group (n = 10), or repaired with the PTFE or 4-HR silk patch in the experimental groups (n = 10 per group). Following patch angioplasty, angiography was performed and the peak systolic velocity (PSV) was measured to evaluate the artery patency. The application of 4-HR was shown to increase the expression of VEGF-A in RAW264.7 and HUVEC cells. The successful artery patency rate was 80% for the 4-HR silk group, 30% for the PTFE group, and 60% for the control group. The PSV of the 4-HR silk group was significantly different from that of the control group at one week and three weeks post-angioplasty (p = 0.005 and 0.024). Histological examination revealed new regeneration of the arterial wall, and that the arterial diameter was well maintained in the 4-HR silk group in the absence of an immune reaction. In contrast, an overgrowth of endothelium was observed in the PTFE group. In this study, the 4-HR silk patch was successfully used as a vascular patch, and achieved a higher vessel patency rate and lower PSV than the PTFE patch.

Activated T cells induce proliferation of oligodendrocyte progenitor cells via release of vascular endothelial cell growth factor-A.

Neuroinflammatory diseases such as multiple sclerosis are characterized by infiltration of lymphocytes into the central nervous system followed by demyelination and axonal degeneration. While evidence suggests that activated T lymphocytes induce neurotoxicity and impair function of neural stem cells, the effect of T cells on oligodendrocyte progenitor cells (OPCs) is still uncertain, partly due to the difficulty in obtaining human OPCs. Here we studied the effect of activated T cells on OPCs using OPCs derived from human hematopoietic stem cells or from human fetal brain. OPCs were exposed to supernatants (sups) from activated T cells. Cell proliferation was determined by EdU incorporation and CellQuanti-Blue assays. Surprisingly, we found that sups from activated T cells induced OPC proliferation by regulating cell cycle progression. Vascular endothelial growth factor A (VEGF-A) transcripts were increased in T cells after activation. Immunodepletion of VEGF-A from activated T cell sups significantly attenuated its effect on OPC proliferation. Furthermore, VEGF receptor 2 (VEGFR2) was expressed on OPCs and its inhibition also attenuated activated T cell-induced OPC proliferation. Thus, activated T cells have a trophic role by promoting OPC proliferation via the VEGFR2 pathway.

Role of alternative splicing of VEGF-A in the development of atherosclerosis.

Vascular endothelial cell growth factor A (VEGF-A) signaling promotes the endothelial cell proliferation, macrophage infiltration and foam cell formation, which play pivotal roles in the pathogenesis of atherosclerosis (AS). However, the role of alternative splicing of VEGF here is not known. Here, ApoE (-/-) mice supplied high-fat diet (HFD mice) were used to generate AS, while ApoE (-/-) mice supplied with normal diet (NOR mice) were used as a control. Aortic endothelial cells (AECs) and infiltrated macrophages were purified and quantified by flow cytometry. Alternative splicing of VEGF and the regulator of VEGF splicing, SRPK1, were assessed by RT-qPCR and immunoblotting in both AECs and aortic macrophages. We found that HFD mice developed AS in 12 weeks, while the NOR did not. Compared to NOR mice, HFD mice possessed significantly more AECs and AEC proliferation, and had significantly more aortic infiltrated macrophages and more apoptosis of them. Significant shift of VEGF-A splicing to pro-angiogenic VEGF165 was detected in both AECs and macrophages from HFD mice, seemingly through upregulation of SRPK1. In vitro, SRPK1 overexpression significantly increased EC proliferation and macrophage apoptosis. Thus, our data suggest that alternative splicing of VEGF-A to pro-angiogenic VEGF165 may contribute to the development of AS.

MicroRNA-638 inhibits cell growth and tubule formation by suppressing VEGFA expression in human Ewing sarcoma cells.

Ewing sarcoma (EWS) is a kind of aggressive tumor of bone and soft tissues, which most occurring in children and adolescents. MicroRNAs (miRNAs) perform essential function in the progression and development of EWS, while the putative role of miR-638 in EWS remains uncertain. Accordingly, we detected the expression of miR-638 and explored its putative biological effects on the malignant phenotype of EWS cells. As expected, miR-638 was significantly down-regulated in EWS cells. Moreover, overexpression of miR-638 suppressed cell growth, induced cell apoptosis, and inhibited tubule formation of EWS cells in vitro. Among the putative target genes of miR-638 predicted by the miRNA target prediction tools, vascular endothelial cell growth factor A (VEGFA) attracted out attention most. The luciferase reporter assays reaffirmed that VEGFA was a targeted gene of miR-638 in EWS cells. Furthermore, miR-638 suppressed the mRNA and protein level of VEGFA, and restored the expression of VEGFA reversed the suppressed effects of miR-638 in EWS cells. Taken together, the results suggested that miR-638 might perform tumor suppressive effects in EWS, which might be mediated, at least partially, through suppressing the activity of VEGFA.

Development of an LC-MS/MS method with protein G purification strategy for quantifying bevacizumab in human plasma.

Biopharmaceutical products such as protein drugs and monoclonal antibodies (mAb) are currently of great interest with monoclonal antibody drugs being one of the fastest growing categories of biopharmaceutical products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained high interest for measuring mAb drugs in biological samples in recent years due to its high selectivity. Bevacizumab is a humanized immunoglobulin G (IgG) mAb drug against human vascular endothelial cell growth factor A (VEGF-A). It is used for treating many types of cancers. Recent studies have indicated that clinical outcomes vary among patients treated with bevacizumab and produce various side effects, such as vascular disorders. In this study, we developed an LC-MS/MS method to quantify bevacizumab concentration. We selected readily available and economic materials for sample preparation to facilitate its wider use in clinical fields.-Protein G was used to trap bevacizumab from human plasma. In place of an extended stable isotope-labeled internal standard (SIL-IS), the IgG-based drug-IS tocilizumab was used because of its better calibration performance. The method was validated in terms of its precision, accuracy, linearity, and sensitivity. The accuracies which were expressed as percentage recoveries for three concentration levels were within 92.8±3.2 to 112.7±4.5%. Repeatability and intermediate precision in terms of peak area ratios were lower than 5.2 and 12.9% RSD, respectively. The application to patients' sample measurements revealed a wide individual variability of drug concentrations, and the proposed simple and general method may facilitate personalized medicine for improving therapeutic efficacy and safety. Graphical abstract ᅟ.

Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells.

Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms.

Role of vascular networks in extending glucose sensor function: Impact of angiogenesis and lymphangiogenesis on continuous glucose monitoring in vivo.

The concept of increased blood vessel (BV) density proximal to glucose sensors implanted in the interstitial tissue increases the accuracy and lifespan of sensors is accepted, despite limited existing experimental data. Interestingly, there is no previous data or even conjecture in the literature on the role of lymphatic vessels (LV) alone, or in combination with BV, in enhancing continuous glucose monitoring (CGM) in vivo. To investigate the impact of inducing vascular networks (BV and LV) at sites of glucose sensor implantation, we utilized adenovirus based local gene therapy of vascular endothelial cell growth factor-A (VEGF-A) to induce vessels at sensor implantation sites. The results of these studies demonstrated that (1) VEGF-A based local gene therapy increases vascular networks (blood vessels and lymphatic vessels) at sites of glucose sensor implantation; and (2) this local increase of vascular networks enhances glucose sensor function in vivo from 7 days to greater than 28 days postsensor implantation. This data provides "proof of concept" for the effective usage of local angiogenic factor (AF) gene therapy in mammalian models in an effort to extend CGM in vivo. It also supports the practice of a variety of viral and nonviral vectors as well as gene products (e.g. anti-inflammatory and anti-fibrosis genes) to engineer "implant friendly tissues" for the usage with implantable glucose sensors as well as other implantable devices.

Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a–Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity

The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We generated and characterized single-chain (sc) fusion proteins of both these domains with linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger protein targeting the vascular endothelial cell growth factor A (VEGF-A) promoter, we demonstrate successful targeting of DNA methylation to the VEGF-A promoter in human cells and observed that almost complete methylation of 12 CpG sites in the gene promoter could be achieved. Targeted methylation by the Dnmt3a-3L sc enzymes was about twofold higher than that of Dnmt3a-C, indicating that Dnmt3a-3L sc variants are more efficient as catalytic modules in chimeric DNA methyltransfeases than Dnmt3a-C. Targeted methylation of the VEGF-A promoter with the Dnmt3a-3L sc variant led to a strong silencing of VEGF-A expression, indicating that the artificial DNA methylation of an endogenous promoter is a powerful strategy to achieve silencing of the corresponding gene in human cells.

Vascular Endothelial Growth Factor A Competitively Inhibits Platelet-Derived Growth Factor (PDGF)-Dependent Activation of PDGF Receptor and Subsequent Signaling Events and Cellular Responses

Certain platelet-derived growth factor (PDGF) isoforms are associated with proliferative vitreoretinopathy (PVR), a sight-threatening complication that develops in a subset of patients recovering from retinal reattachment surgery. Although these PDGF isoforms are abundant in the vitreous of patients and experimental animals with PVR, they make only a minor contribution to activating PDGF receptor α (PDGFRα) and driving experimental PVR. Rather, growth factors outside of the PDGF family are the primary (and indirect) agonists of PDGFRα. These observations beg the question of why vitreal PDGFs fail to activate PDGFRα. We report here that vitreous contains an inhibitor of PDGF-dependent activation of PDGFRα and that a major portion of this inhibitory activity is due to vascular endothelial cell growth factor A (VEGF-A). Furthermore, recombinant VEGF-A competitively blocks PDGF-dependent binding and activation of PDGFR, signaling events, and cellular responses. These findings unveil a previously unappreciated relationship between distant members of the PDGF/VEGF family that may contribute to pathogenesis of a blinding eye disease.

DEP induction of ROS in capillary-like endothelial tubes leads to VEGF-A expression

Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease. One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothelial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified vascular model system for this process, it was previously shown that DEPs induce the redistribution of vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations. This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become permeable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24h of exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth factor-A (VEGF-A), initially termed “vascular permeability factor” (VPF), was found to be up-regulated in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocalization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.

Downstream Exposure to Growth Factors Causes Elevated Velocity and Dilation in Arteriolar Networks

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC₅₀ values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

Endothelial cell activation in a VEGF-A gradient: Relevance to cell fate decisions

Distribution of vascular endothelial cell growth factor A (VEGF-A) as a gradient determines microvascular endothelial cell (EC) fate during organogenesis. While much is understood about mechanisms of differential distribution, less is known about how EC perceive and interpret a graded VEGF-A signal to generate positional target gene activation. Using microvascular EC, we analyzed the effect of time and graded VEGF-A input on VEGFR2 autophosphorylation, signal kinase activation and induction of immediate-early genes. The threshold and time to peak activation of VEGFR2 were dependent on signal strength over a 50-fold range in concentration with 3-fold concentration differences readily distinguished. Longer duration of exposure did not compensate for low concentration of VEGF-A, suggesting intensity and duration of signal were not interpreted equivalently. With the same conditions, graded and time-sensitive information was transduced through the PLCγ/p44/p42MAPK signal pathway but not the parallel AKT pathway. Analysis of MAPK-induced angiogenic immediate-early genes determined that EGR-1, EGR-3, and NR4A1 were dependent on graded input while NR4A2 and DSCR1 were independent with ‘switch-like’ induction. These data demonstrate rapid, linear integration of VEGF-A levels but independent interpretation of duration of signal and identify potential nodes for segregation of gradient-dependent and -independent responses. These results describe how microvascular EC fate decisions can be determined by comparatively moderate changes in VEGF signal strength, resulting in combinatorial changes in the repertoire of immediate-early genes for transcription effectors.