Sphingosine-1-phosphate signaling in vasculogenesis and angiogenesis

Blood vessels either form de novo through the process of vasculogenesis or through angiogenesis that involves the sprouting and proliferation of endothelial cells in pre-existing blood vessels. A complex interactive network of signaling cascades downstream from at least three of the nine known G-protein-coupled sphingosine-1-phosphate (S1P) receptors act as a prime effector of neovascularization that occurs in embryonic development and in association with various pathologies. This review focuses on the current knowledge of the roles of S1P signaling in vasculogenesis and angiogenesis, with particular emphasis on vascular cell adhesion and motility responses.

CREG accelerates vasculogenesis in mouse embryonic stem sellsderived embryonic bodies

Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demonstrated that CREG was expressed in all three germ layers,suggesting that it might act as a vital regulator during embryonic developing.The aim of the present study was to investigate the role of CREG in an embryonic stem cell(ESC) differentiation model that recapitulates the developmental steps of vasculogenesis.Methods The ES cells were stably transfected either pCXN2-FLAG-CREG-IRES-EGFP plasmid or pDS1- shRNA-CREG plasmid to produce the CREG+/ES cells and CREG-siRNA/ES cells,respectively.Vasculogenesis was detected by whole mount immunostainings for CD31.Dil labeled acLDL staining assay was used to detect branching pseudopods in cultures in Matrigel.Real-time PCR and Western blot analysis were employed to determine expressions of VEGF and Flk-1.Results CREG +/ES-derived embryoid bodies(EBs) were found to form spontaneously a primitive vascular network after 6 days of differentiation.In contrast, wildtype EBs exhibit theirs vasculogenesis until 13 days of differentiation by whole mount immunostainings for CD31. CREG +/EBs developed more rapidly branching pseudopods at 9 days compared with that of wildtype EBs by Dil labeled acLDL staining assay.In contrast,CREG-siRNA/ES exhibits an undifferentiated morphogenesis associated with an increase in apoptotic cells in spite of being derived from LIF and feeder layers.Administration of CREG-siRNA/ES cells with recombinant CREG protein rescued the phenomena that CREG boosted vasculogenesis in a dose-dependent fasion. Mechanically,Real-time PCR and Western blot analysis revealed the expressions both VEGF and Flk-1 significantly in- creased in CREG+/EBs.Moreover,after treatment of CREG+ /EBs with neurtralizing antibody against VEGF,the rapid vasculogenesis was significantly repressed.Conclusions Our data strongely demonstrate that CREG play a pivotal role in accelerating vasculogenesis in development of ES cells. VEGF,as its important downstream effector,mediated this bio-function.

Effects of heme oxygenase-1 gene modulated mesenchymal stem cells on vasculogenesis in ischemic swine hearts

Background Mesenchymal stem cells （MSCs） transplantation may partially restore heart function in the treatment of acute myocardial infarction （AMI）. The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 （HO-1） on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells.Methods In vitro, MSCs were divided into four groups： （1） non-transfected MSCs （MSCs group）, （2） MSCs transfected with the pcDNA3.1-Lacz plasmid （Lacz-MSCs group）, （3） MSCs transfected with pcDNA3.1-hHO-1 （HO-1-MSCs group）,and （4） MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin （SnPP）（HO-1-MSCs＋SnPP group）. Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at 〈1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor （TGF-β） and fibroblast growth factor （FGF-2） in the supernatant. In vivo,28 Chinese mini-pigs were randomly allocated to the following treatment groups： （1） control group （saline）, （2）Lacz-MSCs group, （3） HO-1-MSCs group, and （4） HO-1-MSCs ＋ SnPP group. About 1×107 of autologous stem cells or an idertical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging （MRI） assay and postmortem analysis were assessed four weeks after stem cell transplantation.Results Post hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs （（874.88±68.23） pg/ml） compared with Lacz-MSCs （（687.81±57.64） pg/ml, P 〈0.001）. FGF-2 was also significantly increased in the HO-1-MSCs （（1106.48±107.06） pg/ml） compared with the Lacz-MSCs （（853.85±74.44） pg/ml, P 〈0.001 ）.In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group （14.24±1.66/HPFs vs. 11.51±1.34/HPFs, P 〈0.001）. Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group （7.86±2.00/HPFs vs. 6.45±1.74/HPFs, P=0.001）.At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group （（53.17±3.55）% vs. （48.82±2.98）%, P 〈0.05）. However, all these effects were significantly abrogated by SnPP.Conclusion MSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

In vivo investigation on the role of tumor vascular marker CD146 molecule in embryonic vasculogenesis and tumor angiogenesis

CD146 molecule, the surface marker of tumor vascular, plays a key role in the proliferation and migration of vascular endothelial cell. However, due to lack of suitable animal model,

Vascular Channel Formation by Osteosarcoma Cells in Vitro: Vasculogenic Mimicry

Objective: To observe whether there is evidence for vascular channel formation by osteosarcoma cellsin vitro and to illustrate mechanism of vasculogenic mimicry in osteosarcoma.Methods: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed under a phase contrast microscope and an electron microscope.Results: Observation under light microscopy and electron microscopy showed that high aggressive osteosarcoma cells line (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen in the absence of endothelial cells or fibroblasts.Conclusion: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry. Key words osteosarcoma cells line - vasculogenesis mimicry - angiogenesis - 3-dimensional cultures This study was supported in part by the National Natural Sciences Foundation of China (No. 30271314).

Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells

Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.

Proangiogenic Macrophage Development in Human Prenatal Stage:A Key Element in Maternal-Fetal Medicine Puzzle

Introduction Macrophages,a major immune cell type constituting the human innate immune system,are involved in various physiological processes,such as tissue development,remodeling,homeostasis,and repair,crucial for maintaining normal growth and development of embryos/fetuses.1-3 Influenced by their cellular origin and specific tissue environments,macrophages exhibit a diverse range of phenotypes.

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system.In our study,we demonstrated that ethanol of gradient concentra-tions can interfere with the establishment of circulatory system in embryonic zebrafish.The ef-fective concentration to cause 50%malformations(EC_(50))was 182.5 mmol/L.The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol.It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43%treated embryos.We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish.By employing in situ hybridization with endothelial bio-marker(Flk-1),we revealed that ethanol disrupts the establishment of trunk axial vasculature,but has no effect on cranial vessels.Combined with the results of semi-thin histological sections,the in situ hybridization experiments with arterial and venous biomarkers(ephrinB2,ephB4)sug-gested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein.Further study indicated the negative influence of ethanol on the development of hy-pochord in zebrafish.The consequent lack of vasculogenic factors including Radar and Ang-1 partly explains the defects in formation and integrity of dorsal aorta.These results provide im-portant clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.

Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase

SAG(Sensitive to Apoptosis Gene),also known as RBX2(RING box protein 2),ROC2(Regulator of Cullins 2),or RNF7(RING Finger Protein 7),was originally cloned in our laboratory as a redox inducible antioxi-dant protein and later characterized as the second member of the RBX/ROC RING component of the SCF(SKP1-CUL-F-box Proteins)E3 ubiquitin ligase.When acting alone,SAG scavenges oxygen radicals by forming inter-and intra-molecular disulfide bonds,whereas by forming a complex with other components of the SCF E3 ligase,SAG promotes ubiquitination and degradation of a number of protein substrates,includ-ing c-JUN,DEPTOR,HIF-1α,IκBα,NF1,NOXA,p27,and procaspase-3,thus regulating various signaling path-ways and biological processes.Specifically,SAG pro-tects cells from apoptosis,confers radioresistance,and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis.Furthermore,stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis.Finally,SAG transgenic expression in epidermis causes an early stage inhibi-tion,but later stage promotion,of skin tumorigenesis triggered by DMBA/TPA.Given its major role in pro-moting targeted degradation of tumor suppressive proteins,leading to apoptosis suppression and accel-erated tumorigenesis,SAG E3 ligase appears to be an attractive anticancer target.

Progress in tumor vascular normalization for anticancer therapy:challenges and perspectives

Antitumor angiogenic therapy has been shown promising in the treatment of several advanced cancers since the approval of the first antiangiogenic drug Avastin in 2004.Although the current antiangiogenic drugs reduce the density of tumor blood vessels and result in tumor shrinkage at the early stage of treatment,recent studies have shown that antiangiogenic therapy has transient and insufficient efficacy,resulting in tumor recurrence in patients after several months of treatment.Blockage of blood and oxygen supplies creates a hypoxic and acidic microenvironment in the tumor tissues,which fosters tumor cells to become more aggressive and metastatic.In 2001,Jain proposed tumor vascular normalization as an alternative approach to treating cancers based on the pioneering work on tumor blood vessels by several other researchers.At present,normalizing the disorganized tumor vasculature,rather than disrupting or blocking them,has emerged as a new option for anticancer therapy.Preclinical and clinical data have shown that tumor vascular normalization using monoclonal antibodies,proteins,peptides,small molecules,and pericytes resulted in decreased tumor size and reduced metastasis.However,current tumor vascular normalizing drugs display moderate anticancer efficacy.Accumulated data have shown that a variety of vasculogenic/angiogenic tumor cells and genes play important roles in tumor neovascularization,growth,and metastasis.Therefore,multiple-targeting of vasculogenic tumor cells and genes may improve the efficacy of tumor vascular normalization.To this end,the combination of antiangiogenic drugs with tumor vascular normalizing therapeutics,as well as the integration ofWestern medicine with traditional Chinese medicine,may provide a good opportunity for discovering novel tumor vascular normalizing drugs for an effective anticancer therapy.

Infantile hepatic hemangiomas:looking backwards and forwards

Infantile hepatic hemangiomas(IHHs)are common benign tumors seen in the liver of infants.IHHs are true infantile hemangiomas(IHs)and have phases of proliferation and involution parallel to those of cutaneous IHs.The definition and classification of IHH are still confusing in the literature.The mechanisms during the pathogenesis of IHH have yet to be discovered.The clinical manifestations of IHH are heterogeneous.Although most IHH lesions are asymptomatic,some lesions can lead to severe complications,such as hypothyroidism,consumptive coagulopathy,and high-output congestive cardiac failure.Consequently,some patients can possibly encounter a fatal clinical condition.The heterogeneity of the lesions and the occurrence of disease-related comorbidities can make the treatment of IHH challenging.Oral propranolol is emerging as an effective systemic approach to IHH with obvious responses in tumor remission and symptom regression.However,the precise clinical characteristics and treatment strategies for patients with severe IHH have not yet beenwell established.Here,we summarize the epidemiology,pathogenicmechanism,clinical manifestations,diagnosis,and treatment of IHH.Recent updates and future perspectives for IHH will also be elaborated.