Angiogenic Tube Formation Research Articles

Inhibition of GPR4 Attenuates the Formation of Abdominal Aortic Aneurysm Through Inhibiting the SP-1/VEGF-A Signaling.

Abdominal aortic aneurysm (AAA) is a severe cardiovascular disease (CVD) that is partly attributable to endothelial dysfunction, inflammatory response, and angiogenesis. G protein-coupled receptor 4 (GPR4), a proton-sensitive G protein-coupled receptor that is abundantly expressed in vascular endothelial cells, has been associated with numerous physiological functions. Nevertheless, its potential involvement in the development of AAA remains unexplored. In this study, we examined the impact of GPR4 deletion on the development of AAA in ApoE-deficient mice. The mice were categorized into four distinct groups: the ApoE-/- with saline group, the ApoE-/-GPR4-/- with saline group, the ApoE-/- with Ang II group, and the ApoE-/-GPR4-/- with Ang II group. AAA were induced in the ApoE-/- mice through the perfusion of angiotensin II (Ang II). Notably, GPR4 was substantially elevated in the AAA tissues from both human subjects and experimental mice. The deletion of GPR4 substantially decreased the formation of Ang II-induced AAA, damages to elastin, and the expression of aortic inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), as well as vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 (VEGF-A/VEGF-R2), in ApoE-/- mice. Human aortic endothelial cells (HAECs) were transfected with lenti-viral GPR4 shRNA and subsequently stimulated with Ang II. Our findings indicate that the knockout of GPR4 attenuated Ang II-induced angiogenic tube formation in HAECs by decreasing the expression of VEGF-A and VEGF-R2. Furthermore, GPR4 knockout also hindered the activation of specificity protein-1 (SP-1) by reducing its expression and transcriptional activity. Notably, the overexpression of SP-1 reversed the inhibitory effects of GPR4 knockout on angiogenic tube formation and the expression of VEGF-A/VEGF-R2. This suggests that the protective effects of GPR4 knockout are achieved through the inhibition of SP-1. In summary, the absence of GPR4 impeded AAA formation, indicating that GPR4 could potentially serve as a therapeutic target for AAA.

Functional omics of ORP7 in primary endothelial cells

BackgroundMany members of the oxysterol-binding protein-related protein (ORP) family have been characterized in detail over the past decades, but the lipid transport and other functions of ORP7 still remain elusive. What is known about ORP7 points toward an endoplasmic reticulum and plasma membrane-localized protein, which also interacts with GABA type A receptor-associated protein like 2 (GABARAPL2) and unlipidated Microtubule-associated proteins 1A/1B light chain 3B (LC3B), suggesting a further autophagosomal/lysosomal association. Functional roles of ORP7 have been suggested in cholesterol efflux, hypercholesterolemia, and macroautophagy. We performed a hypothesis-free multi-omics analysis of chemical ORP7 inhibition utilizing transcriptomics and lipidomics as well as proximity biotinylation interactomics to characterize ORP7 functions in a primary cell type, human umbilical vein endothelial cells (HUVECs). Moreover, assays on angiogenesis, cholesterol efflux, and lipid droplet quantification were conducted.ResultsPharmacological inhibition of ORP7 leads to an increase in gene expression related to lipid metabolism and inflammation, while genes associated with cell cycle and cell division were downregulated. Lipidomic analysis revealed increases in ceramides and lysophosphatidylcholines as well as saturated and monounsaturated triacylglycerols. Significant decreases were seen in all cholesteryl ester and in some unsaturated triacylglycerol species, compatible with the detected decrease of mean lipid droplet area. Along with the reduced lipid stores, ATP-binding cassette subfamily G member 1 (ABCG1)-mediated cholesterol efflux and angiogenesis decreased. Interactomics revealed an interaction of ORP7 with AKT1, a central metabolic regulator.ConclusionsThe transcriptomics results suggest an increase in prostanoid as well as oxysterol synthesis, which could be related to the observed upregulation of proinflammatory genes. We envision that the defective angiogenesis in HUVECs subjected to ORP7 inhibition could be the result of an unfavorable plasma membrane lipid composition and/or reduced potential for cell division. To conclude, the present study suggests multifaceted functions of ORP7 in lipid homeostasis, angiogenic tube formation, and gene expression of lipid metabolism, inflammation, and cell cycle in primary endothelial cells.

DRIM modulates Src activation and regulates angiogenic functions in vascular endothelial cells.

Downregulated in Metastasis Protein (DRIM) was discovered in malignant epithelial cells and was thought to be mainly a nucleus protein affecting cancer cells. Recent single-cell sequencing analysis suggests that DRIM is abundantly expressed in vascular endothelial cells. There has been no knowledge of the role of DRIM in the endothelium. In the present study, using protein fraction method and cell imaging, we identified that the DRIM protein was abundantly present in both nucleus and the cytoskeletal fractions of human vascular endothelial cells. Knockdown of DRIM in the endothelial cells significantly affected growth, migration, and angiogenic tubule formation. Proteomics analyses revealed that Src was an important direct target protein of DRIM, a finding further confirmed by protein interaction assay. Silencing DRIM activated the tyrosine 419 site phosphorylation of Src kinase in endothelial cells, thereby affecting the downstream proteins of Src including p-FAK and p-STAT3, and exerting biological effects. To conclude, our results provide evidence of DRIM being a nuclear and cytoskeletal-associated protein, having a novel key role of the protein in vascular endothelial cells.

TMIC-41. RBBP4/P300 COMPLEX CONTROLS GENES MODULATING ANGIOGENESIS AND IMMUNOSUPPRESSION IN GLIOBLASTOMA MICROENVIRONMENT

Abstract The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.

H2S-Eluting Hydrogels Promote In Vitro Angiogenesis and Augment In Vivo Ischemic Wound Revascularization.

Ischemic wounds are frequently encountered in clinical practice and may be related to ischemia secondary to diabetes, peripheral artery disease and other chronic conditions. Angiogenesis is critical to the resolution of ischemia. Hydrogen sulfide (H2S) is now recognized as an important factor in this process. H2S donors NaHS and GYY4137 were incorporated into the photosensitive polymer hydrogel gelatin methacrylate and evaluated. Human umbilical vein endothelial cell (HUVEC) culture was used to quantify toxicity and angiogenesis. Sprague Dawley rats were subjected to ischemic myocutaneous flap wound creation with and without application of H2S-eluting hydrogels. Tissue perfusion during wound healing was quantified using laser speckle contrast imaging, and gene and protein expression for VEGF were evaluated. Vascular density was assessed by CD31 immunohistochemistry. Successful incorporation of sulfide compounds was confirmed by scanning electron microscopy with energy-dispersive X-ray analysis, and under physiologic conditions, detectable H2S was present for up to 14 days by high-performance liquid chromatography. HUVECs exposed to hydrogels did not demonstrate excess cytotoxicity or apoptosis. A two-fold increase in angiogenic tube formation was observed in HUVECs exposed to H2S-eluting hydrogels. Rat ischemic flap wounds demonstrated greater perfusion at 14 days, and there was greater vascularity of healed wounds compared to untreated animals. A nearly two-fold increase in VEGF mRNA and a four-fold increase in VEGF protein expression were present in wounds from treated animals. Local-regional administration of H2S represents a novel potential therapeutic strategy to promote angiogenesis and improve wound healing after tissue injury or as a result of ischemic disease.

Abstract WMP118: Utilizing Patient-Derived Induced Pluripotent Stem Cells as a Cellular Model for Studying Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare cerebrovascular disease that can lead to stroke. A histopathological characteristic in MMD is the thickening of the intimal layer that causes narrowing of the vessel, suggesting dysfunctions in the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The mechanism of MMD is largely unknown, due to a lack of established MMD-specific cellular or animal models. In this study, we established patient-derived cellular co-culture models using MMD iPSC-derived ECs and VSMCs. We also generated a 3D vascular organoid model to more closely mimic the MMD vasculature and function. Methods: Peripheral blood mononuclear cells (PBMCs) from MMD patients or healthy controls were used to generate iPSCs (n=4 per group). Differentiated ECs and VSMCs were validated and their functional characteristics were investigated in single and co-cultures. For co-cultures, ECs and VSMCs were labeled with Vibrant cell labeling dyes (DiO and DiD), followed by either transwell migration assay (ECs on bottom of cell culture plate and VSMCs on top Transwell insert) or in vitro angiogenic tube formation assay. Control and MMD iPSCs were used to generate 3D cellular organoids, and their cellular characteristics were examined using immunohistochemistry with antibodies labeling ECs and VSMCs. Results: While single cultures of ECs displayed similar tube formation patterns between control and MMD, co-cultures of ECs with VSMCs showed that MMD ECs failed to stabilize in vitro angiogenic tubes and formed aggregates at 24hr, when compared to control ECs. Transwell migration assays showed MMD VSMCs migrated faster than control VSMCs. Immunostaining of organoids with EC and VSMC markers showed notable differences in cellular composition, with MMD organoid showing compromised integrity of vascular networks, when compared with controls. Conclusions: We have established 3D organoid cellular models and in vitro co-cultures using MMD patient-derived iPSCs. Our co-culture studies revealed functional impairment in MMD EC/VSMC and organoid models revealed distinct characteristics in their vascular networks, possibly contributing to MMD pathology. These results shed useful insight into the cellular and molecular mechanisms driving MMD.

Abstract C116: Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Investigating the effect of vandetanib and celecoxib combination on angiogenesis

ObjectiveAngiogenesis plays an important role in various physiological and pathological conditions and is essential for tumor growth and metastasis. The aim of this study was to evaluate the effect of a combination of vandetanib and celecoxib on angiogenic tube formation and its effect on angiogenic genes (MMP-2 and MMP-9) using an in vitro model of human umbilical vein endothelial cells (HUVECs). MethodsHUVECs were cultured and verified by flow cytometry. HUVECs were then treated with vandetanib, celecoxib, and the combination of both drugs. Then, we investigated cell viability and cell apoptosis by MTT assays and flow cytometry. The process of angiogenesis was analyzed by tube formation assays, and the effect on angiogenic genes was determined by RT-qPCR. ResultsHUVECs were positive for CD144 and negative for CD14. Vandetanib, celecoxib, and their combination inhibited HUVEC viability in a dose-dependent manner (p < 0.001). The rate of apoptosis was 13.1%, 9%, and 23.7% (p < 0.001) when treated with vandetanib, celecoxib, or the combination of both drugs, respectively. Vandetanib inhibited tube formation by 43.7%, celecoxib by 21%, and their combination by 77.3% (p < 0.001), respectively. RT-qPCR revealed that both vandetanib and celecoxib reduced the expression levels of MMP-2 and MMP-9, and their combination resulted in an even greater extent of reduction in expression levels (p < 0.001). ConclusionCelecoxib enhanced the effect of vandetanib in inhibiting in vitro angiogenesis and the combination of these two drugs led to even greater extents of inhibition than vandetanib alone.

Calcium phosphate with submicron topography influences primary human macrophage response, enhancing downstream angiogenesis and osteogenesis in vitro

Calcium phosphates with submicron surface features have demonstrated superior performance to conventional calcium phosphates and equivalence to autologous bone in pre-clinical bone healing models. This is related to their ability to form bone in soft tissues, without the addition of cells and growth factors. It is hypothesized that a specific innate immune response to submicron topography contributes to the enhanced bone healing by these materials. Upregulation of pro-healing, anti-inflammatory ‘M2’ macrophages versus pro-inflammatory ‘M1’ macrophages on submicron-structured calcium phosphates may be involved. In this in vitro study, the response of primary human macrophages to different calcium phosphate bone graft substitutes was assessed. Primary CD14+ monocytes were isolated from human buffy coats and were seeded on two different calcium phosphate materials. The first material had a submicron topography of needle-shaped crystals (BCP<μm) while the second material had no submicron topography (TCP). Macrophage M1/M2 phenotype characterization by protein and gene expression markers at 24 h and 72 h indicated overall stronger macrophage activation and subtle phenotypic skewing towards the M2 phenotype on BCP<μm vs TCP. Moreover, macrophages exhibited an elongated morphology on BCP<μm, which is associated with the M2 phenotype, while macrophages on TCP primarily exhibited a spherical morphology. Conditioned medium of macrophages cultured on BCP<μm resulted in enhanced in vitro angiogenic tube formation and osteogenic differentiation of mesenchymal stromal cells, compared to conditioned medium from macrophages on TCP. Altogether, these findings suggest a potential role of M2 macrophage upregulation in the bone-induction mechanism of calcium phosphates with submicron surface topography.

Injectable anti-cancer drug loaded silk-based hydrogel for the prevention of cancer recurrence and post-lumpectomy tissue regeneration aiding triple-negative breast cancer therapy

A single system capable of delivering anticancer drugs and growth factors by a minimally invasive approach is in demand for effective treatment of triple-negative breast cancer (TNBC) after lumpectomy. Here, we showcase one such holistic system for TNBC therapy and its assessment via 3D in vitro lumpectomy model, a first of its kind. Firstly, Bombyx mori silk fibroin (BMSF) and Antheraea assamensis silk fibroin (AASF) blended hydrogels were prepared and biophysically characterized. Secondly, a 3D in vitro lumpectomy model was developed using MDA-MB-231 cell line to assess the efficacy of localized delivery of doxorubicin (dox) using injectable hydrogel system in terminating remaining breast cancer after lumpectomy. Additionally, we have also evaluated the adipose tissue regeneration in the lumpectomy region by delivering dexamethasone (dex) using injectable hydrogels. Rheological studies showed that the BMSF/AASF blended hydrogels exhibit viscoelasticity and injectability conducive for minimally invasive application. The developed hydrogels by virtue of its slow and sustained release of dox exerted cytotoxicity towards MDA-MB-231 cells assessed through in vitro studies. Further, dex loaded hydrogel supported adipogenic differentiation of adipose tissue derived stem cells (ADSCs), while the secreted factors were found to aid in vascularization and macrophage polarization. This was confirmed through in vitro angiogenic tube formation assay and macrophage polarization study respectively. The corroborated results vouch for potential application of this injectable hydrogels for localized anticancer drug delivery and aiding in breast reconstruction, post lumpectomy.

Peficitinib inhibits fibroblast-like synoviocyte activation and angiogenic vascular endothelial tube formation via inhibitory effects on PDGF and VEGF signaling in addition to JAK

PurposePeficitinib and tofacitinib are known to suppress inflammation in rheumatoid arthritis (RA) by inhibiting Janus kinases (JAKs). However, these effects on tyrosine kinases other than JAKs have not yet been well investigated. We evaluated the effects of peficitinib and tofacitinib on platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) and on the activation of fibroblast-like synoviocytes (FLSs) and endothelial cells, main pathological causes of RA. MethodsPeficitinib and tofacitinib were tested in PDGF and VEGF RTK assays. We then used FLSs derived from RA patient (RA-FLSs) and human umbilical vein endothelial cells (HUVECs) to study the effects of peficitinib and tofacitinib on PDGF- and VEGF-induced signal transduction and on the activation of RA-FLSs and endothelial cell tube formation. FindingsPeficitinib, not tofacitinib, inhibited both PDGF and VEGF RTKs in addition to JAKs in cell-free assay system. Peficitinib and tofacitinib attenuated PDGF- and VEGF-induced intracellular signal transduction pathways in RA-FLSs and HUVECs to varying degrees. Only peficitinib potently inhibited PDGF-induced secretion of interleukin-6, VEGF, and matrix metalloproteinase-3 in RA-FLSs, and endothelial cell tube formation by HUVECs. ConclusionPeficitinib may improve RA through inhibition of PDGF and VEGF signal transduction, in addition to JAK inhibition.

Maternal obesity inhibits placental angiogenesis by down-regulating the SIRT1/PGC-1α pathway.

BackgroundTo explore whether maternal obesity inhibits placental angiogenesis through down-regulation of Sirtuin 1/Peroxisome proliferator-activated receptor-γ coactivator-1α (SIRT1/PGC-1α) signaling pathway.MethodsIn a rat model of pre-pregnancy obesity, rats were sacrificed at embryonic day (E)18.5. Maternal characteristics were measured. Placentas were collected to observe the pathological changes and angiogenesis using hematoxylin-eosin (HE) staining and platelet endothelial cell adhesion molecule-1 [PECAM-1/CD31 (CD31)] immunohistochemical (IHC) staining, and the expression of the SIRT1/PGC-1α signaling pathway was also analyzed using western blotting and quantitative real-time polymerase chain reaction (qPCR). In in vitro experiments, human umbilical vein endothelial cells (HUVECs) were incubated under high fat conditions. We activated and inhibited the SIRT1/PGC-1α signaling pathway to determine the proliferation, angiogenic tube formation, and migration capacity of endothelial cells. Cell counting kit-8 (CCK-8) assays, tubule formation assays, and scratch wound-healing migration assays were also performed.ResultsIn vivo results showed that compared with the control group, the high-fat diet (HFD) group were heavier and their plasma triglyceride and total cholesterol contents were higher. The ratio of fetal weight to placental weight was reduced in the HFD group compared to the control group. In the HFD group, placental angiogenesis was decreased, and the SIRT1/PGC-1α signaling pathway was down-regulated compared with that in the control group. The results of in vitro experiments showed that SRT1720 reduced SIRT1/PGC-1α and vascular endothelial growth factor (VEGFA) expression inhibition induced by high fat stress, while EX-527 increased SIRT1/PGC-1α and VEGFA expression inhibition. Compared with the control group, maternal obesity impaired placental angiogenesis and reduced the proliferation and migration of HUVECs.ConclusionsThe results suggest that maternal obesity impairs placental angiogenesis. They also provide experimental evidence that activation of the SIRT1/PGC-1α signaling pathway improves angiogenesis in vitro.

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.

Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist-Treated Prostate Cancers.

Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.

7, 8-Dihydroxyflavone, a TrkB receptor agonist, provides minimal protection against retinal vascular damage during oxygen-induced ischemic retinopathy.

Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR.

Constitutive Oxidative Stress by SEPHS1 Deficiency Induces Endothelial Cell Dysfunction.

The primary function of selenophosphate synthetase (SEPHS) is to catalyze the synthesis of selenophosphate that serves as a selenium donor during selenocysteine synthesis. In eukaryotes, there are two isoforms of SEPHS (SEPHS1 and SEPHS2). Between these two isoforms, only SEPHS2 is known to contain selenophosphate synthesis activity. To examine the function of SEPHS1 in endothelial cells, we introduced targeted null mutations to the gene for SEPHS1, Sephs1, in cultured mouse 2H11 endothelial cells. SEPHS1 deficiency in 2H11 cells resulted in the accumulation of superoxide and lipid peroxide, and reduction in nitric oxide. Superoxide accumulation in Sephs1-knockout 2H11 cells is due to the induction of xanthine oxidase and NADPH oxidase activity, and due to the decrease in superoxide dismutase 1 (SOD1) and 3 (SOD3). Superoxide accumulation in 2H11 cells also led to the inhibition of cell proliferation and angiogenic tube formation. Sephs1-knockout cells were arrested at G2/M phase and showed increased gamma H2AX foci. Angiogenic dysfunction in Sephs1-knockout cells is mediated by a reduction in nitric oxide and an increase in ROS. This study shows for the first time that superoxide was accumulated by SEPHS1 deficiency, leading to cell dysfunction through DNA damage and inhibition of cell proliferation.

Thalidomide Suppresses Angiogenesis Through the Signal Transducer and Activator of Transcription 3/SP4 Signaling Pathway in the Peritoneal Membrane.

Peritoneal angiogenesis is the key pathophysiological factor that limits peritoneal ultrafiltration during peritoneal dialysis (PD) in uremic patients. Thalidomide has been confirmed to inhibit angiogenesis by inhibiting the secretion of vascular endothelial growth factor (VEGF), but the exact mechanism by which thalidomide inhibits vascular proliferation during PD is still unclear. Here, the objective of the present study was to investigate whether the reduction in VEGF production by human peritoneal mesothelial cells (HPMCs) was controlled by thalidomide. Stimulation of HPMCs with IL-6 in combination with soluble IL-6 receptor (sIL-6R) promoted VEGF expression and secretion, but these effects were attenuated by thalidomide treatment through a transcriptional mechanism that involved signal transducer and activator of transcription 3 (STAT3) and SP4. Conditioned medium from HPMCs cultured with thalidomide inhibited angiogenic endothelial tube formation, which could be further blocked by silencing SP4 and promoted by overexpressing SP4. In vivo, induction of peritoneal angiogenesis in sham rats, sham+PD rats, 5/6 nephrectomy (5/6Nx) rats, 5/6Nx+PD rats, and 5/6Nx+PD rats intraperitoneally treated with thalidomide showed that thalidomide was involved in the control of several key endothelial–specific targets, including VEGFR2, VEGFR3, SP4, and STAT3 expression and new vessel formation, confirming the role of thalidomide and STAT3/SP4 signaling in these processes. Taken together, these findings identify a novel mechanism that links thalidomide, STAT3/SP4 signaling, and angiogenesis in the peritoneal membrane.

Adverse effect of polystyrene microplastics (PS-MPs) on tube formation and viability of human umbilical vein endothelial cells

Environmental contamination by microplastics (MPs) is an emerging concern in recent years due to associated adverse impacts of MPs on potential human health problems. Endothelial dysfunction is a condition in which the endothelial layer fails to form normally, and is associated with impaired vascular function. Despite the fact that MPs are known to enter the circulation system through intestinal epithelium, little has been known whether MPs impact the normal function of endothelial cells and the formation of vasculature. In the current study, we investigated the effect of polystyrene microplastics (PS-MPs) on tube formation and cytotoxicity in human umbilical vein endothelial cells (HUVECs). Our study showed that the treatment of HUVECs with PS-MPs significantly decreased cell viability, with intracellular accumulation occurring in a dose- and size-dependent manner. Moreover, significant dose-dependent inhibition of angiogenic tube formation was observed in HUVECs treated with 0.5 μm PS-MPs; this effect was accompanied by suppression of angiogenic signaling pathways and inhibitory activity against wound healing and cell migration. Regarding the mechanism of decreased viability, we observed increased autophagic and necrotic cell death. These results indicate that 6-h exposure of endothelial cells to PS-MPs represses tube-forming capacity, while 48-h exposure leads to autophagy and necrosis-mediated cytotoxicity.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

Anti‐tumor and Anti‐Angiogenic activity of the Choline Acetyltransferase Inhibitor BW813U, in Human Lung Adenocarcinoma

Lung adenocarcinoma (LACs) accounts for about 60% of all lung cancer cases in the United States. Epidemiological studies show that the development of lung adenocarcinoma is strongly correlated to smoking habits. Although, cigarette smoke is a complex mixture of over 4000 chemicals, nicotine is the active and addictive component of cigarette smoke. Our previous data show that nicotine accelerates the growth of human lung cancers by promoting angiogenesis. The endogenous ligand for nicotine in lung cancer cells is the neurotransmitter ACh. The enzyme choline acetyltransferase (ChAT) synthesizes ACh in mammalian cells. The present study aims to determine the anti-tumor and anti-angiogenic activity of BW813U in human LACs. Human microvascular endothelial cells from the lung (HMEC-L) robustly express enzymatically active ChAT. BW813U robustly blocked the proliferation and angiogenic tubule formation of HMEC-Ls. These experiments were repeated in lung adenocarcinoma-associated endothelial cells (LAAECs) and similar results were obtained. The anti-angiogenic activity of BW813 was confirmed in rat retinal explants assay and chicken chorioallantoic membrane (CAM) assays. Finally, the administration of BW813U potently suppressed the growth of human LAC tumors xenotransplanted in athymic mice. Immunoshistochemical experiments revealed that BW813U-treated human LAC tumors (isolated from athymic mice) contained fewer number of blood vessels than vehicle-treated mice. The anti-angiogenic activity of BW813U was mediated by nicotinic acetylcholine receptors on HMEC-Ls and required the Akt pathway. The administration of BW813U did not cause any discomfort or toxicity in mice. Therefore ChAT blockers (like BW813U) may be useful in suppressing angiogenesis and growth of human LACs.