Proliferation Of Human Vascular Endothelial Cells Research Articles

The water-soluble TF3 component from Eupolyphaga sinensis Walker promotes tibial fracture healing in rats by promoting osteoblast proliferation and angiogenesis

ObjectiveTo determine the active components of Eupolyphaga sinensis Walker (Tu Bie Chong) and explore the mechanisms underlying its fracture-healing ability. MethodsA modified Einhorn method was used to develop a rat tibial fracture model. Progression of bone healing was assessed using radiological methods. Safranin O/fast green and CD31 immunohistochemical staining were performed to evaluate the growth of bone cells and angiogenesis at the fracture site. Methylthiazoletetrazolium blue and wound healing assays were used to analyze cell viability and migration. The Transwell assay was used to explore the invasion capacity of the cells. Tubule formation assays were used to assess the angiogenesis capacity of human vascular endothelial cells (HUVECs). qRT-PCR was used to evaluate the changes in gene transcription levels. ResultsTu Bie Chong fraction 3 (TF3) significantly shortened the fracture healing time in model rats. X-ray results showed that on day 14, fracture healing in the TF3 treatment group was significantly better than that in the control group (P = .0086). Tissue staining showed that cartilage growth and the number of H-shaped blood vessels at the fracture site of the TF3 treatment group were better than those of the control group. In vitro, TF3 significantly promoted the proliferation and wound healing of MC3T3-E1s and HUVECs (all P < .01). Transwell assays showed that TF3 promoted the migration of HUVECs, but inhibited the migration of MC3T3-E1 cells. Tubule formation experiments confirmed that TF3 markedly promoted the ability of vascular endothelial cells to form microtubules. Gene expression analysis revealed that TF3 significantly promoted the expression of VEGFA, SPOCD1, NGF, and NGFR in HUVECs. In MC3T3-E1 cells, the transcript levels of RUNX2 and COL2A1 were significantly elevated following TF3 treatment. ConclusionTF3 promotes fracture healing by promoting bone regeneration associated with the RUNX2 pathway and angiogenesis associated with the VEGFA pathway.

Abstract P1015: Functional Investigation Of The Coronary Artery Disease Risk Gene Rest In Atherosclerosis

Background: GWAS discovered two CAD risk variants, rs17087335 and rs72627509, located downstream of REST (RE1-silencing transcription factor). So far, REST has been mainly associated with neurological diseases, while its role in vascular function remains unclear. Here, we aimed to explore the role of REST in atherosclerosis. Methods and Results: Transcriptome data from the GTEx consortium hinted towards an association of reduced REST expression in vascular tissues with the risk alleles of rs17087335 and rs72627509. We performed reporter gene assays in HEK293 cells using constructs that contain the REST promoter upstream and the SNP sequence (200 bp) downstream of the reporter gene. In line with the GTEx data, the luciferase assays showed reduced reporter gene activity with the risk alleles of both variants. We next studied the function of REST in different atherosclerosis-relevant vascular cell types, such as human vascular smooth muscle cells (HVSMC) and human vascular endothelial cells (HVEC). In HVEC, siRNA-mediated REST downregulation experiments revealed no changes in HVEC proliferation or migration. In HVSMC, in contrast, migration was significantly enhanced secondary to REST knockdown. Furthermore, reduced REST led to increased uptake of lipids into HVSMC under inflammatory conditions. To elucidate putative molecular pathways, we performed RNA sequencing in HVSMC secondary to REST downregulation. Surprisingly, we detected downregulation of ATF6 (ActivatingTranscription Factor 6) upon REST knockdown, which was confirmed by qPCR. In silico analysis revealed several REST binding sites at the ATF6 promoter. ATF6 plays a role in endoplasmatic reticulum (ER) stress, which has been reported to contribute to a macrophage-/fibroblast-like lipid-loaden phenotype of SMC. In line with the findings for REST, we could show that decreased ATF6 results in increased lipid uptake by HVSMC. Conclusion and Outlook: We provide first evidence that reduced REST expression may increase the risk of atherosclerosis and CAD via promoting HVSMC migration and a phenotypic switch towards macrophage-/fibroblast-like lipid-loaden SMC. These effects might be mediated via ATF6 . Modulation of REST might be a new strategy for preventing and treating CAD.

Long non-coding RNA PVT1 regulates atherosclerosis progression via the microRNA-106b-5p/ACSL4 axis

Long non-coding RNAs (lncRNAs) have been associated with atherosclerosis (AS), but the role of lncRNA PVT1 in this disease is still unknown. However, lncRNA PVT1 was found to be significantly upregulated in the serum of AS patients. In vitro experiments using human vascular endothelial cells (HUVECs) showed that oxidized low-density lipoprotein (ox-LDL) treatment enhanced PVT1 expression and impeded HUVEC proliferation, which could be reversed by PVT1 knockdown or miR-106b-5p mimics. Additionally, knockdown of PVT1 and overexpression of miR-106b-5p inhibited the increase of iron content, MDA level, lipid ROS, ACSL4, and PTGS2 in ox-LDL-induced HUVECs, as well as the decrease of GSH and GPX4. We also found that PVT1 knockdown reduced lipid deposition, atherosclerotic plaque number, and size in ApoE−/− mice. These results suggest that PVT1 plays a crucial role in AS progression by regulating the miR-106b-5p/ACSL4 axis in HUVECs, and may therefore be a potential therapeutic target for AS.

Intravitreal injection of EV11, a novel aryl ketone amide, inhibits choroidal neovascularization via AKT/ERK1/2 pathway

PurposeChoroidal neovascularization (CNV) is the major cause of irreversible vision loss associated with age-related macular disease (AMD). The currently clinical chemical therapeutic strategies are of high cost and facing supply chain shortage. In our study, we aim to investigate EV11, a novel derivative from Sorafenib, as a new approach to inhibit the formation of CNV. MethodsCell viability assay, wound healing assay, transwell assay and tube formation assay were applied to explore the effects of EV11 on human vascular endothelial cells (HUVECs). Western blotting analysis was performed to investigate the pathways when EV11 acts on HUVECs. Laser-induced CNV in mice and intravitreal injection of EV11 were applied to find out the efficacy of the drug in vivo. Histological examination and electroretinogram (ERG) evaluated the retinal morphology and visual function after drug application. ResultsEV11 influenced the HUVECs cell viability as the concentration increasing after 24 hour incubation. It influenced HUVECs through suppressing AKT and ERK1/2 pathway. EV11 reduced CNV area with the optimal concentration of 200uM in mice eyes and compared with Bevacizumab, it had the same effect. The retinal thickness around the optic in each group was not influenced. The amplitudes of the a- and b-waves on scotopic and photopic ERG were not reduced after intravitreal injection. ConclusionThe present study indicated that EV11 affected the proliferation, migration and tube formation of HUVECs, inhibited the area of neovascular of laser induced choroidal neovascularization in mice eyes with no toxicity. EV11 could block the AKT/ERK1/2 signaling pathway in effects of HUVECs. This study unveiled a novel perspective drug EV11 to be a potential candidate for neovascularization.

Bioactive Fish Scale Scaffolds with MSCs-Loading for Skin Flap Regeneration.

Skin flap transplantations are common methods for covering and repairing tissue defects in surgery, while the survival rates of these skin flaps are still low due to the vascular crisis and necrosis. To improve this situation, herein a novel biohybrid scaffold is proposed by integrating the advantages of anisotropic fish scales and mesenchymal stem cells (MSCs) for skin flap regeneration. The fish scale scaffold is obtained through its decellularization and decalcification processes, which reserved intact collagen, glycosaminoglycan, and other endogenous growth factors for MSCs and human vascular endothelial cells proliferation. As the scaffold maintains intrinsic anisotropic structures on both surfaces, the proliferative cells can be elongated along the aligned structures on the fish scale, which endow them the capacity to differentiate into multiple directions. Based on these features, it is demonstrated from an in vivo experiment that the MSCs‐loading fish scale scaffolds can effectively convert the activated inflammatory macrophages into anti‐inflammatory properties, reduce the inflammation around the flap, and improve the survival rate. These results indicate that the MSCs‐loading fish scale scaffold is suitable and has the potential for skin flap regeneration and functional recovery.

Artificial Dermal Scaffold Loaded with Platelet-Rich Plasma Promotes Wound Healing in Pigs by Favoring Angiogenesis.

BackgroundThe development of artificial dermis provides a new therapeutic method for full-thickness skin defects. However, the slow regeneration of blood vessels in the wound site still cannot be solved perfectly. In our study, we combined platelet-rich plasma (PRP) with Lando® artificial dermal scaffold to promote vascular regeneration and wound healing in pigs.Material/MethodsFirst, PRP was compounded with the artificial dermal scaffold. Then, this material was co-cultured with human vascular endothelial cells (HUVECs) and the growth and proliferation of HUVECs were assessed. Bama miniature pigs wound models were fabricated, the materials were transplanted into the skin defect, and wound healing and blood vessel regeneration were assessed by HE staining and CD31 immunohistochemistry.ResultsScanning electron microscopy (SEM) showed that PRP formed round particles on the surface of the artificial dermis material. Cell co-culture experiments showed that the PRP composite artificial dermal scaffold can promote the growth and proliferation of HUVECs. CCK8 experiments demonstrated that the number of cells in the PRP composites group on days 2, 3, 4, and 5 was higher than that in the material alone group (P<0.01). The results of animal experiments showed that PRP composite artificial dermal material can promote wound healing. Histological staining and immunohistochemical staining indicated that the PRP composites group promoted epithelial tissue thickening and blood vessel regeneration in wounds (P<0.001).ConclusionsOur experimental results showed that the artificial dermal scaffold loaded with platelet-rich plasma can promote the revascularization of wounds and accelerated wound healing.

Up-regulation of MiR-145-5p promotes the growth and migration in LPS-treated HUVECs through inducing macrophage polarization to M2

MiR-145-5p is high-expressed in human vascular endothelial cells (HUVECs) and alternatively activated macrophages (M2). However, whether miR-145-5p can reduce HUVEC damage by regulating macrophage immunophenotype is less reported. THP-1 was stimulated by Phorbolate-12-myristate-13-acetate, LPS and IFN-γ, and IL-4 to differentiate into macrophages (M0, M1 and M2). The expressions of macrophage markers were detected by Western blotting, and the expressions of miR-145-5p and kruppel-like factor-14 (KLF14) were detected by qRT-PCR. Dual-luciferase reporter assay was used to analyze the targeted relationship of miR-145-5p and KLF14. HUVEC injury was induced by LPS and then co-cultured with M1 transfected by miR-145-5p mimic. The effect of miR-145-3p on proliferation and metastasis of LPS-induced HUVECs was detected by MTT, clone formation, scratch assay and Transwell. We found that the expression of miR-145-5p was higher in M2 than that in M1. MiR-145-5p expression was down-regulated during M2-to-M1, but up-regulated during M1-to-M2. The expressions of IL-1β and iNOS were down-regulated, while the protein expressions of CCL17 and Arg-1 were up-regulated by miR-145-5p mimic in M0. The viability, proliferation, migration and invasion of HUVECs were promoted, however, LDH activity of the HUVECs was inhibited by mimics. In addition, KLF14 was predicted as the target gene for miR-145-5p in HUVECs. Collectively, our results demonstrate that miR-145-5p inhibited cell proliferation of LPS-treated HUVECs possibly through regulating macrophage polarization to M2.

Circular RNA circ_0124644 exacerbates the ox-LDL-induced endothelial injury in human vascular endothelial cells through regulating PAPP-A by acting as a sponge of miR-149-5p.

The modulatory roles of numerous circular RNAs (circRNAs) have been exposited in atherosclerosis (AS). Our study paid attention to the function of circRNA_ 0124644 (circ_0124644) in AS development, as well as its functional mechanism. The AS cell model was established by the treatment of oxidized low-density lipoprotein (ox-LDL) to human vascular endothelial cells (HUVECs). Cell proliferation and cycle were severally measured by Cell Counting Kit-8 (CCK-8) and cell cycle detection kit. The examination of apoptosis rate was executed through flow cytometry. Western blot was exploited for detecting the associated proteins. The expression levels of circ_0124644 and microRNA-149-5p (miR-149-5p) and pregnancy-associated plasma protein-A (PAPP-A) were assayed using quantitative real-time polymerase chain reaction. The combination of targets was validated via the dual-luciferase reporter assay, RNA immunoprecipitation (RIP), and RNA pull-down assay. Clonal capacity was analyzed using colony formation assay. Ox-LDL restrained HUVECs proliferation and cycle, but facilitated apoptosis. Circ_0124644 expression was increased, while miR-149-5p was downregulated in ox-LDL-treated HUVECs. Besides, circ_0124644 served as a molecular sponge of miR-149-5p and intensified the ox-LDL-induced HUVECs injury by sponging miR-149-5p. PAPP-A was a target of miR-149-5p and miR-149-5p could mitigate the HUVECs injury caused by ox-LDL through inhibiting PAPP-A. Moreover, PAPP-A was positively regulated by circ_0124644 via the miR-149-5p. In this report, we concluded the promoted role of circ_0124644 in the ox-LDL-induced endothelial injury of HUVECs via the miR-149-5p/PAPP-A axis with an emphasis on its diagnostic and therapeutic values in AS.

CircRNA‑0044073 is upregulated in atherosclerosis and increases the proliferation and invasion of cells by targeting miR‑107.

Circular RNAs (circRNAs) are endogenous non‑coding RNAs implicated in atherosclerosis. The aim of the present study was to explore the function of circRNA‑0044073 in atherosclerosis. Reverse transcription quantitative polymerase chain reaction assays were used to measure the expression levels of circRNA‑0044073, microRNA (miRNA/miR)‑107, janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), B‑cell lymphoma 2 (Bcl‑2) and v‑myc avian myelocytomatosis viral oncogene homolog (c‑myc) in in blood cells from patients with atherosclerosis. RNA pull‑down and luciferase reporter assays were then used to determine the association between circRNA and miR expression, and miR and gene expression, respectively. Matrigel invasion assay and flow cytometry were used to analyze cell invasion and cell cycle. Western blot analysis and ELISA were used to evaluate the expression levels of proteins. It was identified that the expression of circRNA‑0044073 was upregulated and the expression of miR‑107 was downregulated in atherosclerotic blood cells. Overexpression of circRNA‑0044073 promoted the proliferation of human vascular smooth muscle cells (HUVSMCs) and human vascular endothelial cells (HUVECs), while overexpression of miR‑107 inhibited their proliferation. In addition, circRNA‑0044073 suppressed the levels of miR‑107 via a sponge mechanism. Lipopolysaccharide (LPS) affected the proliferation of HUVSMCs and HUVECs, and also resulted in changes in circRNA‑0044073 expression levels. CircRNA‑0044073 promoted the proliferation and invasion of HUVSMCs and HUVECs in spite of the opposite effect observed with LPS treatment. The JAK/STAT signaling pathway was activated in patients with atherosclerosis. CircRNA‑0044073 favored the activation of the JAK/STAT signaling pathway and inflammation in HUVSMCs and HUVECs. These data indicate that circRNA‑0044073 is upregulated in atherosclerosis and promotes the proliferation and invasion of cells by targeting miR‑107 and activating the JAK/STAT signaling pathway, potentially offering a target for novel treatment strategies against atherosclerosis.

Interaction of Poly(l-lysine)/Polysaccharide Complex Nanoparticles with Human Vascular Endothelial Cells.

Short TitlePolyelectrolyte nanoparticles and vascular endothelial cells.Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.

Hyaluronic acid and α-elastin based hydrogel for three dimensional culture of vascular endothelial cells

The aim of this work was to demonstrate that employing a copolymer of hyaluronic acid and α-elastin (HA-EDA-g-α-elastin) is possible to produce a scaffold able to support the adhesion and growth of human vascular endothelial cells (HUVEC) thanks to its ability to incorporate and control the diffusion of vascular endothelial growth factor (VEGF).HA-EDA-g-α-elastin was crosslinked with low molecular weight hyaluronic acid (HALMW) to obtain a hydrogel that gives rise to the formation of three dimensional sponge after the freeze drying process. The physicochemical features the obtained material along with its ability to act as a support for the three dimensional culture of endothelial cells were here investigated.The obtained hydrogel is able to seize the VEGF and control its release in the first days of incubation. The presence of the growth factor stimulates the proliferation of HUVEC and induces the formation of tubular structures.

The effects of recombinant human platelet derived growth factor-BB on biological behaviours of human retinal vascular endothelial cells

Objective This study was to investigate the role of recombinant human platelet derived growth factor-BB(rhPDGF-BB) in the proliferation and migration of human retinal vascular endothelial cells (hRVECs). Methods hRVECs were cultured in DMEM with 10% fetal bovine serum.The rhPDGF-BB at the concentrations of 10, 50 and 200 ng/ml were added into the medium of exponential phase-growth cells for 24 and 48 hours, respectively, and no rhPDGF-BB was added in the normal control group.The proliferation of the cells (absorbancy) was assayed by cell counting kit 8(CCK8) method.Cell scratch test was employed to evaluate the relative migration area of cells (migrated acellular area/initial acellular area). The relative expression of rhPDGF-BB recepter (rhPDGF-BBR) mRNA in the cells was detected by reverse transcription PCR.The relative expression of VEGF mRNA and integrin mRNA in the cells was detected using real-time fluorescence quantitative PCR. Results hRVECs grew well and a expressing band according with rhPDGF-BBR prime was displayed.The absorbancy values of the cells were 1.01±0.05, 1.09±0.04, 1.10±0.02 and 1.13±0.05 in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups at 24 hours after culture, and those in the 10, 50 and 200 ng/ml rhPDGF-BB groups were significantly increased in comparison with the normal control group (t=2.504, 3.430, 3.483, all at P<0.05). The relative migrated areas of the cells in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups were 0.42±0.10, 0.38±0.09, 0.55±0.06 and 0.61±0.05 at 24 hours after culture, and those at 48 hours were 0.75±0.06, 0.81±0.02, 0.87±0.02 and 0.98±0.02, showing significant differences among the groups (Fgroup=16.283, P=0.000; Ftime=209.129, P=0.000), and the relative migrated areas was depended upon the rhPDGF-BB dose and time.The relative expressions of integrin mRNA were 1.06±0.02, 1.30±0.10, 1.20±0.16 and 1.27±0.08, and those of VEGF mRNA were 0.97±0.05, 1.06±0.16, 1.58±0.18 and 1.66±0.21 in the normal control group and 10, 50 ng/ml, 200 ng/ml rhPDGF-BB groups, respectively, and increased expressions of integrin mRNA and VEGF mRNA were found in the 50 and 200 ng/ml rhPDGF-BB groups compared with the normal control group (integrin mRNA: t=3.900, 4.014, both at P<0.05; VEGF mRNA: t=6.940, 7.210, both at P< 0.05). Conclusions rhPDGF-BB/rhPDGF-BBR signal promotes the proliferation and migration of hRVECs probably by up-regulating the expressions of integrin and VEGF. Key words: Endothelium, vascular/cytology; Neovascularization, pathologic/metabolism; Platelet-derived growth factor; Receptors, platelet-derived growth factor; Recombinant proteins; Humans; Cells, cultured; Integrin; Vascular endothelial growth factor

Abstract B19: Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer

Abstract Background: Tumor requires excessive oxygen and nutrients due to their rapid growth. Tumor is generally vascular but the vasculature is not physiologic but leaky, dilated, and tortuous. The inefficiency of tumor vessels induces hypoxic stress that makes the tumor more aggressive. Here, we investigated the role of normalizing tumor vasculature on tumor progression and treatment efficacy of conventional chemotherapy in prostate cancer. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of SAC-1004 on endothelial cells. Immunofluorescent confocal imaging with immunocytochemistry was performed for E-cadherin expression in HUVECs. MTS assay was performed to assess cell proliferation of HUVECs and PC-3 cells after SAC-1004 treatment. Immunohistochemical staining with anti-CD31 and anti-SMA antibodies was performed to assess vasculature in vivo. PC-3 subcutaneous xenografts were developed to evaluate the effects of SAC-1004 on tumor vasculature and tumor growth in prostate cancer. Magnetic resonance images were taken to assess vascular perfusion in xenografts. SAC-1004 was administrated via tail vein once a day for 7 days. Docetaxel was administrated intravenously once a week. Results: SAC-1004 restored junction protein E-cadherin in vitro in endothelial cells and significantly reduced VEGF-induced retinal vascular leakage. SAC-1004 promoted proliferation of HUVECs in vitro in a dose-dependent manner. However, proliferation of cancer cells was not significantly affected by SAC-1004 treatment. Reduced vascular leakiness and improved hypoxia were found in PC-3 xenografts treated with SAC-1004 treatment and magnetic resonance imaging after SAC-1004 treatment for 7 days confirmed improved perfusion in the PC-3 xenografts treated with SAC-1004 compared with control. Interestingly, SAC-1004 treatment suppressed tumor growth in PC-3 xenografts but SAC-1004 followed by intravenous docetaxel treatment showed the most potent tumor suppression compared with control in PC-3 xenografts. Conclusions: SAC-1004 improves hypoxic tumor microenvironment and restores abnormal leak vasculature of tumor by restoring endothelial cell junction in prostate cancer. Vascular normalization induced tumor regression and docetaxel combination following vascular normalization sensitized prostate cancer to docetaxel treatment. Citation Format: You Hyun Kang, Hyun A Jin, Jun Hyeok Heo, Ki Hong Kim, Kyung Seok Han, Sung Joon Hong. Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B19.

Abstract B18: CXCL10 suppresses tumor angiogenesis and impedes expression of critical angiogenic factors in renal cell carcinoma

Abstract Background: Angiogenic progress is complex and several factors are involved as a promoter or inhibitor of angiogenesis. Tumor can use the imbalance of pro- and antiangiogenic factors in progression. The CXC chemokine family are consisted of pro- or antiangiogenic factors. Recent studies revealed CXCL10 as an anti-angiogenic factor in response to angiogenic factors like bFGF or VEGF. However, a role of CXCL10 in cancer and angiogenesis is unclear. Here, we investigated the role of CXCL10 on tumor angiogenesis and its mechanisms in renal cell carcinoma. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of CXCL10 on endothelial cells. MTS assay was performed to assess cell proliferation. Cell cycle analysis was performed using flow cytometry with propidium iodide. RT-PCR and western blot were performed to assess the baseline expression of CXCL10 in a panel of renal cell carcinoma cells. Overexpression of CXCL10 was developed by lentiviral transfection. Quantitative RT-PCR for angiogenic factors (VEGF, PDGF, FGF, MMP9) was performed by angiogenesis multigene screening array. Subcutaneous tumor xenografts were developed using Caki-1 cells to evaluate in vivo effects of CXCL10. Results: CXCL10 inhibited proliferation and tube formation of HUVEC in a dose-dependent manner, while it did not affect in vitro tumor proliferation of renal cell carcinoma cells. Expression of CXCL10 was downregulated in Caki-1, UMRC3 and UMRC6 cells. Overexpression of CXCL10 suppressed expression of angiogenic factors including VEGF-A, PDGF, FGF2 and MMP9 in Caki-1 cells, suggesting that CXCL10 is involved non only as an anti-angiogenic factor but also as a suppressor of critical angiogenic factors in renal cell carcinoma. In vivo Restoration of CXCL10 expression suppresses tumor growth in renal cell carcinoma by suppressing tumor angiogenesis in xenografts. Conclusions: CXCL10 has a role as an anti-angiogenic factor and also as a suppressor of critical angiogenic factors in renal cell carcinoma. Restoration of CXCL10 expression suppresses tumor growth in renal cell carcinoma by impeding expression of other angiogenic factors including VEGF, PDGF, FGF, MMP9 in renal cell carcinoma as well as inducing anti-angiogenic effect on endothelial cells. Our data suggest that targeting CXCL10 might be a novel therapeutic option for advanced renal cell carcinoma. Citation Format: Hyun A Jin, Jun Hyeok Heo, You Hyun Kang, Ki Hong Kim, Kyung Suk Han, Sung Joon Hong. CXCL10 suppresses tumor angiogenesis and impedes expression of critical angiogenic factors in renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B18.

Abstract B12: Endothelial stabilizer SAC-1004 normalizes tumor vasculature and potentiates docetaxel chemotherapy in prostate cancer

Abstract Background: Since tumor requires excessive oxygen and nutrients due to their rapid growth, tumor is generally vascular. However, tumor vasculature is not physiologic but leaky, dilated, and tortuous. The inefficiency of tumor vessels in terms of perfusion induces hypoxic stress that makes the tumor more aggressive. Here, we investigated the role of a novel endothelial stabilizer SAC-1004 on tumor vascular normalization and its effects on tumor growth in prostate cancer. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of SAC-1004 on endothelial cells. Immunocytochemical staining and immunofluorescent confocal imaging was performed for E-cadherin expression in HUVECs. MTS assay was performed to assess cell proliferation of HUVECs and PC-3 cells after SAC-1004 treatment. Immunohistochemical staining with anti-CD31 and anti-SMA antibodies was performed to assess vasculature in vivo. PC-3 subcutaneous xenografts were developed to evaluate the effects of SAC-1004 on tumor vasculature and tumor growth in prostate cancer. Magnetic resonance images were taken to assess vascular perfusion in xenografts. SAC-1004 was administrated via tail vein once a day for 7 days. Docetaxel was administrated intravenously once a week. Results: SAC-1004 restored junction protein E-cadherin in vitro in endothelial cells and significantly reduced VEGF-induced retinal vascular leakage. SAC-1004 promoted proliferation of HUVECs in vitro in a dose-dependent manner. However, proliferation of cancer cells was not significantly affected by SAC-1004 treatment. Reduced vascular leakiness was found in PC-3 xenografts treated with SAC-1004 treatment and magnetic resonance imaging after SAC-1004 treatment for 7 days confirmed improved perfusion in the PC-3 xenografts treated with SAC-1004 compared with control. Interestingly, SAC-1004 treatment suppressed tumor growth in PC-3 xenografts but SAC-1004 followed by intravenous docetaxel treatment showed the most potent tumor suppression compared with control in PC-3 xenografts. Conclusions: SAC-1004 restored abnormal leak vasculature of tumor by restoring endothelial cell junction in prostate cancer. Vascular normalization by SAC-1004 induced tumor regression and combination with docetaxel following vascular normalization potentiated anti-tumor effects of docetaxel in prostate cancer. Citation Format: Yoo Hyun Kang, Jun Hyeok Heo, Hyun A Jin, Ki Chung Park, Sung Joon Hong, Kyung Seok Han. Endothelial stabilizer SAC-1004 normalizes tumor vasculature and potentiates docetaxel chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B12.

In vitro and ex vivo hemocompatibility of off-the-shelf modified poly(vinyl alcohol) vascular grafts

Synthetic small diameter vascular grafts with mechanical properties of native arteries, resistance to thrombosis and capacity to stimulate in situ endothelialization are an unmet clinical need. Poly(vinyl alcohol) hydrogel (PVA) is an excellent candidate as a vascular graft due to its tunable mechanical properties. However, the hydrophilicity and bio-inertness of PVA prevents endothelialization in vivo. We hypothesize that the modification of PVA with biomolecules and topographies creates a hemocompatible environment that also enhances bioactivity. PVA modified with fibronectin, RGDS peptide, cyclicRGD (cRGD) peptide, or heparin provided cell-adhesion motifs, which were confirmed by detection of nitrogen through X-ray photoelectron spectroscopy. Protein- and peptide-modified surfaces showed a slight increase in human vascular endothelial cell proliferation over unmodified PVA. With the exception of fibronectin modification, modified surfaces showed in vitro hemocompatibility comparable with unmodified PVA. To further improve bioactivity, cRGD–PVA was combined with gratings and microlens topographies. Combined modifications of 2μm gratings or convex topography and cRGD significantly improved human vascular endothelial cell viability on PVA. In vitro hemocompatibility testing showed that topography on cRGD–PVA did not significantly trigger an increase of platelet adhesion or activation compared with unpatterned PVA. Using the more physiologically relevant ex vivo hemocompatibility testing, all PVA grafts tested showed similar platelet adhesion to ePTFE and significantly lower platelet accumulation compared to collagen-coated ePTFE grafts. The biochemical and topographical modification of PVA demonstrates excellent hemocompatibility with enhanced bioactivity of PVA, thus highlighting its potential as a vascular graft. Statement of SignificanceNew synthetic small diameter vascular grafts with mechanical properties, blood-clot resistance and endothelial lining mimicking native arteries remains an unresolved critical clinical need. We aim to achieve this by modifying the mechanically-tunable poly(vinyl alcohol) hydrogel (PVA) vascular graft with both biochemical and biophysical cues in the lumenal surface. PVA modified with cyclic RGD peptide and ordered micrometer-sized topography showed low platelet adhesion in both a rabbit in vitro and baboon ex vivo blood compatibility assay. Modified PVA also exhibited significant enhancement of human vascular endothelial cell viability and proliferation in vitro. The readily available, modified PVA grafts are the first to show biophysical and biochemical modification in a three-dimensional scaffold with hemocompatibility, biofunctionality and excellent potential for clinical application.

Clopidogrel reduces apoptosis and promotes proliferation of human vascular endothelial cells induced by palmitic acid via suppression of the long non-coding RNA HIF1A-AS1 in vitro.

Cardiovascular disease (CVD) is recognized as a major and increasing health problem affected older subjects in China, and clopidogrel has been widely used for treatment of CVD patients such as atherosclerosis, myocardial infarction, and myocardial ischaemia-reperfusion damage. However, the molecular mechanisms of clopidogrel for treatment of CVD are only partially understood. This study investigated the effects of clopidogrel on palmitic acid-induced damage of human vascular endothelial cells (HUVECs), and the molecular mechanisms of LncRNA HIF1A-AS1 in regulating the proliferation and apoptosis of HUVECs in vitro. We firstly established a damage model of HUVECs through palmitic acid (PA) treatment. And the effect of clopidogrel reducing PA-induced apoptosis of HUVECs was observed by the flow cytometric measurement. To further understand the molecular mechanism of clopidogrel rescues PA-induced apoptosis, we used human LncRNA PCR array to compare the LncRNA expression profile difference between clopidogrel-treated cells and control cells. The expression of LncRNA HIF 1 alpha-antisense RNA 1 (HIF1A-AS1) was significantly altered in clopidogrel-treated cells. We further proved that suppression of HIF1A-AS1 by siRNA reduce PA-induced apoptosis and promote proliferation of HUVECs. Furthermore, we also demonstrated inhibition apoptosis effect by HIF1A-AS1 is related to mitochondrial apoptosis pathway. Hence, our results suggest that clopidogrel rescues apoptosis and promotes proliferation of PA-induced damage model of HUVECs through inhibiting the mediator LncRNA HIF1A-AS1. These findings indicate that LncRNA HIF1A-AS1 may play an important role in the pathogenesis of CVD, and provide a novel molecular mechanism of clopidogrel for treatment of CVD.

Preparation of topographically modified poly(L-lactic acid)-b-Poly(ɛ-caprolactone)-b-poly(L-lactic acid) tri-block copolymer film surfaces and its blood compatibility

Hindering early platelet adhesion and enhancing human vascular endothelial cell proliferation on surfaces of the foreign materials play a key role in the prevention of coagulation. In order to develop topographically suitable surface for artificial grafts, topographically modified poly(L-lactic acid)-b-poly(ɛ-caprolactone)-b-poly(L-lactic acid) tri-block copolymer (PLLA-PCL-PLLA) film surfaces were cast using a simple solvent-nonsolvent method. PLLA-PCL-PLLA copolymer was synthesized, and was confirmed by 1H nuclear magnetic resonance (NMR) analysis. The molecular weight of the copolymer was measured using gel permeation chromatography (GPC). PLLAPCL-PLLA films were cast with various ratios of non-solvent in the solvent mixture. Tetrahydrofuran (THF) and ethyl alcohol (EtOH) were used as solvent and nonsolvent respectively. The hydrophobic characteristic of the surfaces was confirmed by the water contact angle (WCA). The prepared film surfaces were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffraction (XRD). Anti-platelet adhesion characteristic was evaluated using Lactate Dehydrogenase (LDH) assay and SEM images. Proliferation behavior of human vascular endothelial cell was investigated by water-soluble tatrazolium salt (WST) assay after 3 h, 1 day, 2 days and 4 days of cell culture on the film surface. Fabricated morphologically unique surface, which consists of submicron size width and nano size height rough structure, shows anti platelet adhesive ability and relatively higher human vascular endothelial cell proliferation behavior.

Urea immunoliposome inhibits human vascular endothelial cell proliferation for hemangioma treatment

BackgroundUrea injection has been used in hemangioma treatment as sclerotherapy. It shrinks vascular endothelial cells and induces degeneration, necrosis, and fibrosis. However, this treatment still has disadvantages, such as lacking targeting and difficulty in controlling the urea dosage. Thus, we designed a urea immunoliposome to improve the efficiency of treatment.MethodsThe urea liposome was prepared by reverse phase evaporation. Furthermore, the urea immunoliposome was generated by coupling the urea liposome with a vascular endothelial growth factor receptor (VEGFR) monoclonal antibody using the glutaraldehyde cross-linking method. The influence of the urea immunoliposome on cultured human hemangioma vascular endothelial cells was observed preliminarily.ResultsUrea immunoliposomes showed typical liposome morphology under a transmission electron microscope, with an encapsulation percentage of 54.4% and a coupling rate of 36.84% for anti-VEGFR. Treatment with the urea immunoliposome significantly inhibited the proliferation of hemangioma vascular endothelial cells (HVECs) in a time- and dose-dependent manner.ConclusionsThe urea immunoliposome that we developed distinctly and persistently inhibited the proliferation of HVECs and is expected to be used in clinical hemangioma treatment.

T-helper cell type 2 (Th2) memory T cell-potentiating cytokine IL-25 has the potential to promote angiogenesis in asthma

IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25(+), IL-25R(+), and CD31(+) (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25(+), IL-25R(+), and CD31(+)/IL-25R(+) cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25(+) cells correlated inversely with the forced expiratory volume in 1 s (r = -0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25-induced angiogenesis, and the inhibitors also reduced IL-25-induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.