Capillary Formation Assay Research Articles

High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression.

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high‐mobility group box‐1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC‐associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34‐positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1‐treated or HMGB1‐TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti‐HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin‐8 (IL‐8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1‐TF were significantly reversed by IL‐8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL‐8 mediation, and combined targeting of HMGB1 and IL‐8 can control tumor angiogenesis in GC.

2K1C-activated Angiotensin II (Ang II) exacerbates vascular damage in a rat model of arthritis through the ATR/ERK1/2 signaling pathway.

To explore the role and mechanism of the two-kidney one-clip (2K1C)-activated Angiotensin II (Ang II) in the development of vascular damage in adjuvant-induced arthritis (AA) rats. 2K1C rats were established in normal and AA rats for 35days. Hypertension, endothelial dysfunction, and vascular hypertrophy induced by 2K1C-activated Ang II in systemic inflammation rats were evaluated. The levels of Ang II and TNF-α in serum were observed by ELISA kits. Expressions of Ang II/ATR/ERK1/2 signaling pathway molecules in the aorta were tested by immunohistochemistry or western blot. The migration and capillary tube formation abilities of human umbilical vein endothelial cells (HUVECs) were tested by migration chamber and capillary tube formation assays. The level of Ang II in serum was significantly increased in 2K1C rats. Compared with AA rats, the high level of Ang II activated by 2K1C reduced the endothelium-dependent vasodilator responses to acetylcholine (ACh) in the thoracic aorta and exacerbated endothelial dysfunction and vascular hypertrophy. Expressions of ATR, GRK2, p-ERK1/2, and p-NF-κB were significantly increased in the aorta of AA combined with 2K1C rats. The migration and capillary tube formation abilities of HUVECs were significantly enhanced by Ang II and TNF-α co-stimulations in vitro through the ATR/ERK1/2 signaling pathway compared to those stimulated with TNF-α. 2K1C-activated Ang II is involved in aggravated vascular injury and endothelial dysfunction through the ATR/ERK1/2 signaling pathway in AA rats.

Resveratrol suppresses pulmonary tumor metastasis by inhibiting platelet-mediated angiogenic responses

BackgroundTo explore the impact of Resveratrol (RSV) on the angiogenic potential of activated platelets and to elucidate the underlying mechanism. MethodsVascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay. Capillary tube formation assay was used to examine the impact of RSV on the angiogenic potential of activated platelets. The levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) in the supernatant were evaluated using corresponding enzyme-linked immunosorbent assay kits. Immunoblotting assays were used to determine the expression of vasodilator-stimulated phosphoprotein and Akt phosphorylation. A pulmonary metastasis experiment with male nude mice model was performed to test the effect of RSV on pulmonary metastasis and angiogenesis in vivo. ResultsRSV inhibited platelets-mediated angiogenic responses induced by adenosine diphosphate (ADP)ADP through increased cGMP generation and cGMP-mediated vasodilator-stimulated phosphoprotein phosphorylation along with reduced intracellular Ca2+ mobilization. In addition, RSV attenuated the platelet secretion and angiogenic responses induced by A549 cells in vitro and suppressed A549 lung cancer metastasis and angiogenesis in nude mice. ConclusionsRSV is a potential therapeutic drug for the prevention of tumor metastasis by interrupting the platelet-tumor cell amplification loop.

Abstract P211: N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) Promotes Tube Formation in Human Coronary Artery Endothelial Cells

Ac-SDKP is an endogenous peptide with anti-inflammation and anti-fibrotic effects in hypertensive and cardiovascular diseases. It is cleaved from Thymosin β4 (Tβ4) and hydrolyzed by angiotensin converting enzyme (ACE). Ac-SDKP plasma concentration increases after treatment with ACE inhibitors (ACEi) and some of the beneficial effects of ACEi treatment has been ascribed to Ac-SDKP. Ac-SDKP is a mediator of angiogenesis in in-vitro and in-vivo animal models. Ac-SDKP stimulates rodents derived immortalized aortic endothelial cells migration and capillary-like structures formation (tube formation). Similarly, Ac-SDKP increases capillary density after myocardial infarction in rats. The mechanism related to angiogenesis induced by Ac-SDKP is not known. Tβ4 (Ac-SDKP precursor) promotes endothelial cell migration and angiogenesis by the activation of the VEGF/AKT pathway. Our objective is to evaluate the Ac-SDKP pro-angiogenic effect in Human Coronary Artery Endothelial Cells (HCAEC) and the mechanism that regulates the angiogenic effect of Ac-SDKP. HCAEC do not produce VEGF, thus we hypothesize that Ac-SDKP increases VEGF expression in fibroblasts and that indirectly could promote capillary tube formation in endothelial cells. We used primary culture of rat cardiac fibroblast (RCF) and we treated these cells with 10nM Ac-SDKP for 24 hours. VEGF concentration in cell supernatant was measured by ELISA. Cells were starved without serum overnight before the Ac-SDKP treatment. For capillary tube formation assay, HCAEC cells were seeded into matrigel and incubated in presence of 10nM Ac-SDKP for 12 hours, pictures were taken by double phase contrast microscope and tube length was quantified with image J software and the results were expressed as percentage of control. After Ac-SDKP treatment, VEGF concentration did not increase in the supernatant of RCF (control: 0.12±0.07 vs. Ac-SDKP: 0.14±0.09 mg/ml; p=0.7). However, Ac-SDKP treatment induced the development of tube formation in HCAECs by 7±2% respect to control (p=0.037). We conclude that Ac-SDKP induces capillary tube formation not only in rodent but also in human derived endothelial cells. The mechanism by which Ac-SDKP promotes tube formation in HCAECs is still unknown.

Activated hepatic stellate cells promote angiogenesis via interleukin-8 in hepatocellular carcinoma.

BackgroundChemokines have been recognized as important modulators of angiogenesis, and they play critical roles in the development and metastasis of hepatocellular carcinoma (HCC), although their origins and latent molecular mechanisms remain elusive. The aim of this study was to investigate how activated hepatic stellate cells (a-HSCs) promote angiogenesis in HCC.MethodsA total of 22 HCC patients were enrolled randomly. We used immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA) to analyse the production of interleukin-8 (IL-8) in a-HSCs derived from HCC tissues. The angiogenic effects of IL-8 in vitro and in vivo were assessed by ELISA, real-time quantitative polymerase chain reaction, capillary tube formation assay, and chick embryo chorioallantoic membrane assay.ResultsThe present study showed that IL-8 was enriched predominantly in the tumour stroma of HCC tissues and was mainly derived from a-HSCs, rather than from hepatoma cells, in vivo and in vitro. Angiogenesis was most active at the invading edge, which was close to the a-HSCs. The angiogenic effect was dramatically attenuated by an IL-8 neutralizing antibody both in vitro and in vivo. Moreover, the IL-8 neutralizing antibody down-regulated Ser727-phosphorylated STAT3 levels in hepatoma cells treated with a-HSCs conditioned medium.ConclusionsThese findings reveal that a-HSCs within the stroma of HCC contribute to tumour angiogenesis via IL-8.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0730-7) contains supplementary material, which is available to authorized users.

The differentiation of hMSCs counteracts their migration capability and pro-angiogenic effects in vitro.

Human mesenchymal stem cells (hMSCs) have been applied therapeutically in numerous clinical trials. The pro-angiogenic effects of hMSCs, as well as their strong tumor tropism, have been shown both in vitro and in vivo. Some studies suggest using hMSCs as potential drug-carriers for tumor therapy. In previous investigations by our group, the pro-tumorigenic effects of hMSCs on head and neck squamous cell carcinoma (HNSCC) were shown. However, the influence of hMSCs on tumor vascularization as well as the possibility of its inhibition are yet to be elucidated. The cytokine patterns of the HNSCC cell line FaDu, native hMSCs (hMSCs-nat), hMSCs differentiated into adipocytes (hMSCs-adip) and osteocytes (hMSCs-ost) were evaluated. Human umbilical vein endothelial cells (HUVECs) were co-cultured with FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost. The capillary tube formation assay was applied. Furthermore, the migration capability of hMSCs-nat, hMSCs-adip and hMSCs-ost towards FaDu cells was measured in a Transwell system. Spheroids were cultured from hMSCs-nat, FaDu cells and DiI-labeled HUVECs. FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost released a wide range of cytokines and growth factors, e.g., IL-6, IL-8, IL-10, GRO and MCP. In the capillary tube formation assay, HUVECs generated significantly longer tubes after co-cultivation with hMSCs-nat as compared to HUVECs alone and FaDu. Differentiation into adipocytes and osteocytes counteracted the tube formation. The adipogenic differentiation did not alter hMSC motility, whereas osteogenic differentiation significantly inhibited hMSC migration. Generation of multi-cellular spheroids from hMSCs-nat, FaDu cells and DiI-labeled HUVECs was possible. Florescence microscopy revealed that all HUVECs were present in the spheroid core. Taken together, hMSCs-nat have a pro-angiogenic effect. The effects are counteracted by the differentiation of hMSCs towards osteogenic and adipogenic lineages. The differentiation of stem cells into different lineages may be a promising solution to generating carriers for cancer therapy without pro-tumorigenic properties.

Combined Effects of Growth Hormone and Mineral Trioxide Aggregate on Growth, Differentiation, and Angiogenesis in Human Dental Pulp Cells

IntroductionThe aim of this study was to evaluate the effects of growth hormone (GH) on mineral trioxide aggregate (MTA) with regard to cell adhesion, growth, odontoblastic differentiation, and angiogenesis in human dental pulp cells and the underlying signal pathway mechanisms. MethodsCell adhesion and proliferation were assessed by adhesion analysis and cell counting. Differentiation was examined by alkaline phosphatase activity, alizarin red staining, and reverse transcriptase polymerase chain reaction for marker genes. Angiogenesis was evaluated by human umbilical vein endothelial cell migration and capillary tube formation assays. Signaling pathways were analyzed by Western blotting and confocal microscopy. ResultsCombined treatment with GH and MTA enhanced cell adhesion, growth, alkaline phosphatase activity, calcified nodules, expression of marker mRNAs, migration, and capillary tube formation, compared with treatment with MTA or GH alone. In addition, GH plus MTA increased expression of bone morphogenetic protein-2 mRNA, phosphorylation of Smad 1/5/8, extracellular signal-regulated kinase, JNK, and p38 MAPK, and increased the levels of the transcription factors Runx2 and Osterix, compared with MTA alone. ConclusionsCollectively, our results demonstrate that a combination of MTA and GH promotes cell adhesion, growth, differentiation, and angiogenesis of MTA in human dental pulp cells via the activation of bone morphogenetic protein and MAPK pathway.

P53 suppresses cell proliferation, metastasis, and angiogenesis of osteosarcoma through inhibition of the PI3K/AKT/mTOR pathway

ObjectiveTo investigate the role of P53 in the pathogenesis of osteosarcoma and the possible mechanism involved in it. MethodsThe anti-proliferative effect of P53 was assessed using the cell counting Kit-8 assay. The migration and invasion potential were analyzed using wound-healing and transwell assays, respectively. The Matrigel capillary tube formation assay was performed to mimic in-vivo angiogenesis. Immunoblotting and immunofluorescence were used to observe protein levels and distribution of actin fibers. Finally, S2448p-mammalian target of rapamycin (mTOR) expression was detected on osteosarcoma tissues using immunohistochemistry. ResultsFirstly, P53 potently inhibited cell proliferation in osteosarcoma cell line (MG63) and in human normal osteoblasts (hFOB1.19) in vitro at the IC50 ranged from 50 to 500 nmol/l. Then, an inhibitory effect of P53 on metastasis was observed in osteosarcoma cell line MG63, along with the cytoskeletal rearrangements and suppression of the phosphorylation of PI3K downstream factors including AKT and mTOR. ConclusionThese results show that P53 suppresses cell proliferation and angiogenesis of osteosarcoma through inhibition of the PI3K/AKT/mTOR pathway, which might be an effective novel therapeutic candidate against osteosarcoma in the future.

PP242 suppresses cell proliferation, metastasis, and angiogenesis of gastric cancer through inhibition of the PI3K/AKT/mTOR pathway.

Although preclinical work with rapalogs suggests potential in the treatment of gastric cancer, they have been less successful clinically. In this study, we report the impact of the investigational drug PP242, a potent and selective small-molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis. The antiproliferative effect of PP242 was assessed using the Cell Counting Kit-8 assay. The migration and invasion potential were analyzed using wound-healing and transwell assays, respectively. The Matrigel capillary tube formation assay was performed to mimic in-vivo angiogenesis. Immunoblotting and immunofluorescence were used to observe protein levels and distribution of actin fibers. Finally, p-mammalian target of rapamycin (mTOR) expression was detected on gastric cancer tissues using immunohistochemistry. First, PP242 potently inhibited cell proliferation in gastric cancer cell lines and in human endothelial cells in vitro at the IC50 ranged from 50 to 500 nmol/l. Then, an inhibitory effect of PP242 on metastasis was observed in gastric cancer cell AGS, along with the cytoskeletal rearrangements and suppression of the phosphorylation of PI3K downstream factors including AKT, mTOR, and P70S6K. Furthermore, PP242 was found to decrease the tube formation and migration of human umbilical vein endothelial cells. Using immunohistochemistry, we found that p-mTOR staining was observed in 41.8% (82/196) of gastric cancer tissues and correlated with depth of mural invasion, lymph node metastasis, tumor node metastasis stage, and vascular invasion. These results show that PP242 suppresses cell proliferation and angiogenesis of gastric cancer through inhibition of the PI3K/AKT/mTOR pathway, which might be an effective novel therapeutic candidate against gastric cancer in the future.

Effects of three-dimensional culture on cell phenotype and hematopoietic cytokine expression of umbilical cord mesenchymal stem cells

This study was aimed to investigate the change of cell phenotype and the expression of hematopoiesis associated cytokines in umbilical cord mesenchymal stem cells (UC-MSC) in three-dimensional (3-D) system. MSC were isolated from umbilical cord, and then cultured in 2-D and 3-D system respectively. The phenotype of MSC was detected by flow cytometry; the angiogenic capability of MSC cultured in 2-D and 3-D syitem was assessed using in vitro capillary formation assay. The cytokine expression of MSC in two kinds of culture conditions was measured by real-time PCR. The results showed that MSC were successfully isolated from umbilical cord. Flow cytometry showed that the percentage of CD31, CD133 and CD271 expressed in endothelial cells, endothelial progenitor cells and primitive mesenchymal stem cells increased significantly in 3-D culture conditions, as compared to 2-D system. Capillary formation assay showed that the angiogenic capability of UC-MSC was greatly enhanced. Quantitative PCR showed that the expression of β-actin was upregulated in 3-D system. The expression of some cytokines associated with hematopoiesis, such as G-CSF, LIF, SCF, IL-1α, IL-1β, IL-3, IL-7 and IL-11, increased, especially for LIF, IL-3, IL-7. The expression of IL-10 associated with immune regulation also increased. The expression of SDF-1, IL-6 slightly decreased, but without significant difference. It is concluded that expression of CD31, CD133 and CD271 increases in 3-D system, the angiogenic capability of UC-MSC enhances and the expression of hematopoiesis-associated cytokines in UC-MSC increases in 3-D system.

Abstract 1095: microRNA-26a expression may suppress tumor growth by modulating macrophage infiltration in tumor through down-regulation of M-CSF in hepatocellular carcinoma

Abstract Purpose To Study molecular changes specific to tumor-derived endothelial cells. Materials and methods Tumor tissue and adjacent nontumor tissue were obtained from 27 hepatocellular carcinoma (HCC) patients. Primary cultured CD31+ and CD105+ tumor endothelial cells (TECs) and nontumor endothelial cells (NECs) were isolated by magnetic-activated cell sorting. Affymetrix GeneChip Human Gene ST Arrays were used to determine gene expression profiles of HCC cells and matched TECs and NECs. A capillary tube formation assay was used to evaluate tube formation by the endothelial cells. The tumorigenicity of isolated TECs was monitored in nonobese diabetic/severe combined immunodeficient mice. Results The isolated CD31+ and CD105+ TECs and NECs displayed features of endothelial cells. Both cultured HCC cells and TECs induced capillary tube formation in vitro and were incorporated into tumor vasculature in vivo. The TEC isolates had a gene signature resembling that of HCC cells, and TEC-specific genes were associated with poorer HCC survival. Conclusions A subset of TECs may originate from tumor cells, and they express a unique set of genes linked to HCC prognosis. Note: This abstract was not presented at the meeting. Citation Format: Zong-Tao Chai, Xiao-Dong Zhu, Jian-Yang Ao, Ling-Qun Kong, Yuan-Yuan Zhang, Hui-Chuan Sun. microRNA-26a expression may suppress tumor growth by modulating macrophage infiltration in tumor through down-regulation of M-CSF in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1095. doi:10.1158/1538-7445.AM2014-1095

MiR-126-3p suppresses tumor metastasis and angiogenesis of hepatocellular carcinoma by targeting LRP6 and PIK3R2.

BackgroundThe deregulation of microRNAs has been reported to play a pivotal role in hepatocellular carcinoma (HCC). MiR-126-3p has been reported to be associated with poor prognosis in HCC. However the underlying mechanism of miR-126-3p in HCC remains unclear.MethodsThe expression levels of miR-126-3p in HCC tissues and cells were detected by RT-PCR. Transwell assay and capillary tube formation assay were applied to assess the metastasis and angiogenesis in vitro. Nude mice subcutaneous tumor model was used to perform in vivo study. Dual- luciferase reporter assay was conducted to confirm the direct binding of miR-126-3p and target genes. The changes of biomarker protein levels were examined by western blot and Immunohistochemistry.ResultsWe observed that the miR-126-3p expression levels in HCC tissues and cells were significantly down-regulated. Through gain- and loss- of function studies, we showed that miR-126-3p dramatically inhibited HCC cells from migrating and invading extracellular matrix gel and suppressed capillary tube formation of endothelial cells in vitro. Furthermore, overexpression of miR-126-3p significantly reduced the volume of tumor and microvessel density in vivo. LRP6 and PIK3R2 were identified as targets of miR-126-3p. Silencing LRP6 and PIK3R2 had similar effects of miR-126-3p restoration on metastasis and angiogenesis individually in HCC cells. Furthermore, the miR-126-3p level was inversely correlated with LRP6 and PIK3R2 in HCC tissues. In addition, the rescue experiments indicated that the metastasis and angiogenesis functions of miR-126-3p were mediated by LRP6 and PIK3R2.ConclusionOur results demonstrates that deregulation of miR-126-3p contributes to metastasis and angiogenesis in HCC. The restoration of miR-126-3p expression may be a promising strategy for HCC therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0259-1) contains supplementary material, which is available to authorized users.

An effective method of isolating endothelial cells from intact rat aorta.

Rat aorta has been widely utilized in studies on cardiovascular diseases. Due to its small size, the endothelial cells are difficult to isolate. Here, we developed a modified method to obtain the endothelial cells from the rat aorta. The thoracic aorta of Wistar rat was harvested with its inside was turned out to expose the tunica intima. With its both ends ligated and cultured in ECM in T25 flask, a small amount of cells grew out from the aorta on day 4 and manifested the characteristic cobble stone-like monolayer on day 9-10. The cells were identified to be endothelial cells using reverse transcription (RT)-polymerase chain reaction (PCR), immunocytochemical staining for Von Willebrand Factor (vWF), Dil-Ac-LDL uptake assay, and capillary tube formation assay. This method serves as an easy way to get a large amount of endothelial cells from small-sized vessels, which, therefore, will greatly benefit the researches of vascular disease.

Net platelet angiogenic activity (NPAA) correlates with progression and prognosis of non-small cell lung cancer.

Circulating platelets are abundant sources of angiogensis molecules for the tumor vasculature affecting tumor growth and metastasis. The relationship between non-small cell lung cancer (NSCLC) and intra-platelet levels of VEGF, TSP-1 and net platelet angiogenic activity (NPAA) is unclear. The aim of this study was to better understand the role of these factors in the progression of NSCLC cancer and to assess its clinical significance. Platelet VEGF and TSP-1 and NPAA were measured preoperatively in 68 patients with NSCLC by ELISA or Capillary tube formation assay. VEGF, TSP-1 and NPAA distributions in cancer patients and healthy volunteers were compared using the Mann-Whitney U test. The Kaplan-Meier method, univariate and multivariate regression analysis was used to analyze the correlation between these factors and clinicopathological features, overall survival and disease-free survival. Mean intra-platelet TSP-1 level was slightly higher in patients than in healthy subjects (p = 0.092). Intra-platelet TSP-1 levels were significantly higher in patients with involvement greater than T2 or stage III, compared to other patients. Mean intra-platelet VEGF level was 40.8 pg/106 in patients compared to 21.9 ng/106 in healthy subjects (p = 0.041). Median value of NPAA in patients was significantly higher than that in healthy controls (p<0.001). Patients with high NPAA are more likely to exhibit aggressive clinical pathological features. NPAA greater than the median are associated with poor prognosis. The elevated NPAA have better correlation with tumor microvessel density (MVD) than platelet-derived VEGF. The areas under receiver operating curve (AUROC) of NPAA were higher than that of platelet derived VEGF in different groups. A multivariate analysis showed that NPAA are independent prognostic factors. These results indicated that NPAA may be a clinically useful indicator for diagnostic and prognostic evaluation in NSCLC patients.

Genetically Engineered Myoblast Sheet for Therapeutic Angiogenesis

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.

Oncogenic Insult In Bone Marrow Endothelial Cells Causes Hematopoietic Abnormalities

Bone marrow endothelial cells (BMECs), the cells forming bone marrow vasculature, have been implicated as a critical constituent of bone marrow microenvironment essential for the maintenance of normal and malignant hematopoietic stem cells. They are a part of BM niche regulating the self-renewal and differentiation of hematopoietic stem cells (HSCs) and the homing and progression of pre-leukemia and leukemia cells. It has been realized that pathological angiogenesis such as increased bone marrow microvessel density is associated with a spectrum of human diseases including MDS, MPD and leukemia. Recent discovery of oncogenic mutations of BCR-ABL and JAK2V617F in BMECs and spleen ECs primes the hypothesis that pathological angiogenesis from oncogenic insults may promote or facilitate abnormal hematopoiesis leading to the development and maintenance of blood malignancies. Clinical studies have linked germline mutations of Ras signaling components (e.g. NF1, SHP2, CBL and K-RAS) with JMML, a subgroup of MPD/MDS that is difficult to cure and for which bone marrow transplantation often results in relapses and graft failure. Studies in animal models have shown that activation of oncogenic K-Ras in hematopoietic stem cells could partially recapitulate the symptoms of these MPD/MDS patients, suggesting a HSC intrinsic role of oncogenic transformation. It remains to be seen if deregulated K-Ras signaling in the BM microenvironment, BMECs in particular, may also contribute to the hematologic abnormalities and disease progression, and whether pathologicial BMECs provide an addition therapeutic opportunity to limit transplant-associated relapse. To this end, we recently established a new mouse model that allows endothelial lineage-specific, inducible genetic manipulation of BMECs in adult animals in vivo and a cell culture system to select, expand and functional assay freshly isolated murine BMEC activities in vitro. A strain of Tie2-CreER transgenic mice crossed with loxP-stop-loxP fluorescence reporters (EGFP or Td-tomato) is sensitive to Tamoxifen induction in specifically turning on Cre activity in endothelial cells in 4-8 week old animals with over 80% efficiency in BMECs. Our culture conditions select and expand mouse primary BMECs in a large scale without immortalization or genetic manipulation, and the resulting BMECs retain endothelial lineage markers and are fully functional in 2-demensional and 3-demensional capillary formation assays as well as Matrigel implant plug assay. Using this inducible model, we generated Tie2CreER/LSL-K-Ras G12D mice, to specifically turn on K-Ras G12D in endothelial cells to test if an oncogenic K-Ras insult in EC compartment can affect hematopoiesis. Three consecutive Tamoxifen injections to the mice readily activated K-Ras G12D expression in the BMECs, but not in the blood lineages. The mice bearing endothelial K-Ras G12D died 4-6 months after Tamoxifen induction. An analysis of the K-Ras G12D mice revealed that the oncogenic insult caused a significant change in BM vascular structure and abnormal hematopoiesis. K-Ras G12D mutation in the ECs led to pathological angiogenesis in the bone marrow as manifested in the K-Ras G12D mice an increased colony-forming unit EPC activity, drastically increased vessel permeability, and increased BMEC proliferation. The pathological angiogenesis in the bone marrow was associated with abnormal blood phenotypes in K-Ras G12D mice, showing a significant increase of WBC and lymphocyte counts in peripheral blood, splenomegaly, severe anemia, and a significant increase of CFU-C activity in the blood and spleen. Ongoing transplant experiments will address whether the oncogenic K-Ras expression in endothelial cells is sufficient to fully promote blood cell transformation. In parallel, the oncogenic K-Ras expressing BMECs in primary culture are being examined to determine whether abnormal Ras signaling including RAF-MEK and/or PI3K-AKT pathways are involved in mediating BMEC proliferation and angiogenesis. Our study suggests a novel concept that pathologic bone marrow microenvironment, BMECs in particular, can play an important role in blood cell transformation, and implies that rational targeting of oncogenic pathways in the BMECs may provide an additional therapeutic avenue for blood malignancies, besides direct suppression of abnormal hematopoietic cells, to restore normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Stromal cell–derived factor-1 promoted angiogenesis and inflammatory cell infiltration in aneurysm walls

A small percentage of cerebral aneurysms rupture, but when they do, the effects are devastating. Current management of unruptured aneurysms consists of surgery, endovascular treatment, or watchful waiting. If the biology of how aneurysms grow and rupture were better known, a novel drug could be developed to prevent unruptured aneurysms from rupturing. Ruptured cerebral aneurysms are characterized by inflammation-mediated wall remodeling. The authors studied the role of stromal cell-derived factor-1 (SDF-1) in inflammation-mediated wall remodeling in cerebral aneurysms. Human aneurysms, murine carotid artery aneurysms, and murine intracranial aneurysms were studied using immunohistochemistry. Flow cytometry analysis was performed on blood from mice developing carotid or intracranial aneurysms. The effect of SDF-1 on endothelial cells and macrophages was studied by chemotaxis cell migration assay and capillary tube formation assay. Anti-SDF-1 blocking antibody was given to mice and compared with control (vehicle)-administered mice for its effects on the walls of carotid aneurysms and the development of intracranial aneurysms. Human aneurysms, murine carotid aneurysms, and murine intracranial aneurysms all expressed SDF-1, and mice with developing carotid or intracranial aneurysms had increased progenitor cells expressing CXCR4, the receptor for SDF-1 (p < 0.01 and p < 0.001, respectively). Human aneurysms and murine carotid aneurysms had endothelial cells, macrophages, and capillaries in the walls of the aneurysms, and the presence of capillaries in the walls of human aneurysms was associated with the presence of macrophages (p = 0.01). Stromal cell-derived factor-1 promoted endothelial cell and macrophage migration (p < 0.01 for each), and promoted capillary tube formation (p < 0.001). When mice were given anti-SDF-1 blocking antibody, there was a significant reduction in endothelial cells (p < 0.05), capillaries (p < 0.05), and cell proliferation (p < 0.05) in the aneurysm wall. Mice given anti-SDF-1 blocking antibody developed significantly fewer intracranial aneurysms (33% vs 89% in mice given control immunoglobulin G, respectively; p < 0.05). These data suggest SDF-1 is associated with angiogenesis and inflammatory cell migration and proliferation in the walls of aneurysms, and may have a role in the development of intracranial aneurysms.

Microrna-195 Suppresses Angiogenesis and Metastasis of Hepatocellular Carcinoma By Inhibiting the Expression of VEGF, VAV2, and CDC42

Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down-regulation of miR-195 expression in HCC tissues. In this study, the role of miR-195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR-195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence-free survival. Both gain-of-function and loss-of-function studies of in vitro models revealed that miR-195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR-195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR-195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR-195 phenocopied the effects of miR-195 restoration, whereas overexpression of these targets antagonized the function of miR-195. Furthermore, we revealed that miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR-195 down-regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. miR-195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR-195 expression may be a promising strategy for HCC therapy.

Automated Image Analysis of In Vitro Angiogenesis Assay

Angiogenesis is the biological process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth, and metastasis). In vitro angiogenesis assays provide a platform for evaluating the effects of pro- or antiangiogenic compounds. One of the most informative assays is the endothelial cells capillary tube formation assay performed on a biological matrix. This assay is based on quantification of the stimulatory and inhibitory effects of various agents, which is estimated through the measurement of the pseudo-tubules network length. This standard measurement is usually carried out manually by trained operators but requires time, attention, and dedication to achieve a reasonable degree of accuracy. Moreover, the screening is operator dependent. In this article, we propose an automated procedure to evaluate the pseudo-tubule network lengths. We propose a series of image analysis procedures developed using a freely available image analysis software library. More than 800 images from 12 experiments were analyzed automatically and manually, and their results were compared to improve and validate the proposed image analysis procedure. The resulting image analysis software is currently running on a dedicated server, with comparable accuracy to manual measurements. Using this new automated procedure, we are able to treat 540 images, or three complete assays per hour.

Identification of colonic metabolites of 5‐hydroxylnobiletin and their roles in colon cancer inhibition

Previously, we reported that oral administration of 5‐ hydroxylnobiletin (5HN, a citrus flavonoid) potently inhibited colon carcinogenesis in mice. To further understand the molecular mechanism involved, we investigated colonic metabolism of 5HN in mice after oral administration for 16 weeks. Utilizing HPLC, UV spectroscopy, 1H and 13C NMR, and mass spectroscopy, we identified 8 metabolites of 5HN in the colon. They are mono‐, di‐, or tri‐demethylated derivatives of 5HN. We synthesized 3 major metabolites i.e. 5,3′‐didemethylnobiletin (M1), 5,4′‐ didemethylnobiletin (M2), and 5,3′,4′‐tridemethylnobiletin (M3), and compared their bioactivities with those of 5HN. All 3 metabolites showed stronger anti‐proliferative and pro‐apoptotic effects on colon cancer cells than 5HN, especially M1 was more than 50‐fold stronger than 5HN. M1 showed similar antiinflammatory effects as 5HN in lipopolysaccharide‐stimulated macrophages, while M2 was 3‐fold more potent in anti‐inflammation than 5HN. Moreover, M1 showed more than 10‐fold stronger anti‐angiogenic activities than 5HN in a capillary tube formation assay of human umbilical vein endothelial cells. In conclusion, our results demonstrated that colonic metabolites of 5HN had potent activities in inhibiting cancer cell growth, suppressing inflammation, and decreasing angiogenesis, which may contribute significantly to colon cancer inhibition.Grant Funding Source: NIH, AICR and USDA