Expression Of Endothelial Marker Genes Research Articles

A new 3D-printed polylactic acid-bioglass composite for bone tissue engineering induces angiogenesis in vitro and in ovo.

Large bone defects such as those that occur after trauma or resections due to cancer still are a challenge for surgeons. Main challenge in this area is to find a suitable alternative to the gold-standard therapy, which is highly risky, and a promising option is to use biomaterials manufactured by 3D printing. In former studies, we demonstrated that the combination of polylactic acid (PLA) and bioglass (BG) resulted in a stable 3D-printable material, and porous and finely structured scaffolds were printed. These scaffolds exhibited osteogenic and anti-inflammatory properties. This 3D-printed material fulfills most of the requirements described in the diamond concept of bone healing. However, the question remains as to whether it also meets the requirements concerning angiogenesis. Therefore, the aim of this study was to analyze the effects of the 3D-printed PLA-BG composite material on angiogenesis. In vitro analyses with human umbilical vein endothelial cells (HUVECs) showed a positive effect of increasing BG content on viability and gene expression of endothelial markers. This positive effect was confirmed by an enhanced vascular formation analyzed by Matrigel assay and chicken chorioallantoic membrane (CAM) assay. In this work, we demonstrated the angiogenic efficiency of a 3D-printed PLA-BG composite material. Recalling the osteogenic potential of this material demonstrated in former work, we manufactured a mechanically stable, 3D-printable, osteogenic and angiogenic material, which could be used for bone tissue engineering.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Angiogenesis In Vitro and In Ovo

Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration.

Exploring the transcriptomic network of multi-ligand scavenger receptor Stabilin-1- and Stabilin-2-deficient liver sinusoidal endothelial cells.

The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-β/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood.

Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum-collagen hydrogels.

Lack of bone grafts appeals for bone augmentation solutions. We aimed at osteogenic differentiation of human adipose stem cells (hASCs) and microvascularization in coculture with human umbilical vein endothelial cells (HUVECs) embedded in three-dimensional (3D) gellan gum (GG) and collagen type I (COL) hydrogel mixture. We compared endothelial growth medium-2 (EGM-2) and bioactive glass extract-based endothelial and osteogenic medium (BaG EM-OM) for vascularized bone-like graft development in vitro. Cell viability, cell number, and osteogenic and endothelial gene expression were analyzed. Mineralized hydroxyapatite residues, immunocytochemical staining of endothelial marker CD31 production and late osteogenic marker osteocalcin were imaged. With both media, good cell viability was observed within 3D hydrogel. EGM-2 condition induced significantly higher cell number compared to BaG EM-OM condition at both 7 and 14 days. Interestingly, both media supported osteogenic as well as endothelial marker gene expression. Moreover, formation of reticulated cellular structures was observed in both EGM-2 and BaG EM-OM conditions. However, hydroxyapatite mineralization and strong osteocalcin staining were detected only in BaG EM-OM condition. Importantly, strong production of CD31 and elongated tube-like structures were apparent in EGM-2 culture alone. In conclusion, we demonstrated efficient hASC osteogenic differentiation and microvessel-like network formation in coculture with HUVECs.

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment.

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1F92A ) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.

SU31 - ESTABLISHING A BLOOD-BRAIN BARRIER MODEL FOR THE ANALYSIS OF ALZHEIMER'S DISEASE USING PATIENT DERIVED INDUCED PLURIPOTENT STEM CELLS

Background Alzheimer's Disease (AD) is the most common form of dementia. One characteristic of AD is the accumulation of amyloid-β (Aβ) peptides in different regions of the brain caused by the sequential cleavage of the transmembrane amyloid precursor protein. These extracellular amyloid plaques are a result of a dysregulated Blood-Brain Barrier (BBB) and emerge either due to an augmented production or a decreased excretion through brain barriers. The BBB with its several transporter systems shows age dependent expression levels of unspecific Aβ transporters. Recently, 19 loci including ABCA7, an ABC transporter responsible for Aβ efflux at the BBB, were discovered showing genome-wide significant association with the sporadic, late-onset form of AD (LOAD). Together with other research groups we identified several mutations in ABCA7 in AD patients, which is why an analysis of mutated ABCA7 in AD-related BBB models should be considered. We established patient-specific pluripotent stem cells from AD patients and healthy donors to differentiate endothelial cells and other related BBB cells for the investigation of disease mechanisms related to ABCA7 in AD patients. Methods Episomal vectors were used for the generation of induced Pluripotent Stem (iPS) cell lines from AD-specific B-lymphoblastoid cell lines (B-LCLs) and control B-LCLs of healthy donors. Alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers verified pluripotency. Successfully established lines were applied for the generation of BBB cells and neighboring cells. Endothelial cells and astrocytes were characterized by transcript analysis and flow cytometry. The differentiation process was analyzed for the altered expression of ABCA7. Results AD-specific iPS cells were successfully generated from B-LCLs. We could show alkaline phosphatase activity and the nuclear localization of pluripotency-related transcription factors including OCT4, SOX2, and NANOG. Transcript analysis and western blot analysis verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The efficient differentiation of high quality endothelial cells was shown by the expression of endothelial marker genes including von Willebrand factor. Astrocyte-specific markers including EAAT1 demonstrated efficient astrocyte differentiation. Discussion Human iPS cells provide a powerful tool for the analysis of AD-associated and patient-specific mutations in ABCA7. We successfully established endothelial cells and astrocytes representing suitable to study disease mechanisms of the BBB. Accordingly, this approach is an excellent opportunity to analyze pathogenic phenotypes of AD in vitro towards the identification of potential therapeutic targets.

Atheroprone flow enhances the endothelial-to-mesenchymal transition.

The endothelial-to-mesenchymal transition (EndoMT) is a cellular process featuring decreased expression of endothelial marker genes but increased expression of mesenchymal marker genes. The EndoMT is involved in endothelial dysfunction and the pathogenesis of atherosclerosis. To investigate the dynamic expression of EndoMT genes in vascular endothelial cells under atheroprotective pulsatile shear stress (PS) and atheroprone oscillatory shear stress (OS), we analyzed RNA sequencing data from multitimepoint shear-stress experiments. This unbiased analysis involving next-generation sequencing confirmed that PS and OS had an opposite effect in regulating EndoMT genes. Further experimental validations with H2O2 and gain- and loss-of-function approaches indicated that reactive oxygen species are involved in OS-induced EndoMT, whereas AMP-activated protein kinase and sirtuin-1 could inhibit OS-induced EndoMT. Furthermore, compared with PS, OS increased the DNA methylation of the promoter regions of von Willebrand factor, CD31, and cadherin 5 genes but decreased that of cadherin 2, fibroblast-specific protein 1, and vimentin. The translational implication of the present study builds on the ability of the antidiabetic drug metformin and cholesterol-lowering drug atorvastatin to suppress the EndoMT in cultured endothelial cells and in mouse aortas. NEW & NOTEWORTHY Our RNA sequencing data provided a genome-wide and unbiased view of the shear stress regulation of the endothelial-to-mesenchymal transition (EndoMT) in the endothelium. Furthermore, epigenetic regulation (e.g., DNA methylation) is a key mechanism involved in shear stress-regulated EndoMT. The translational implication of this study is that cardiovascular medications such as statins and metformin have similar beneficial effects as that of atheroprotective flow by mitigating EndoMT.

Vegf signaling promotes vascular endothelial differentiation by modulating etv2 expression

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.

Abstract 13863: PGC-1α Mediates Differentiation Arrest of Murine Bone Marrow-derived Endothelial Progenitor Cells in Diabetes

Introduction: Endothelial progenitor cells (EPCs) in murine bone marrow lineage-negative c-kit+Sca-1+ (BM-KSL) fraction are mobilized in circulation upon tissue injury to mediate a neovascularization process called vasculogenesis, which requires proper differentiative capacity of EPCs. Diabetics suffer defective vasculogenesis, a substantial mechanism for diabetic angiopathy in ischemic limb and heart. How it occurs, however, remains largely unknown. We recently demonstrated that type 1 (T1) and T2 diabetes (DM) induces a transcriptional coactivator PGC-1α in endothelial cells (ECs), and that EC PGC-1α mediates impaired cell migration and angiogenesis in DM. PGC-1α was also found to increase in EPCs cultivated from T2DM patients PB-MNCs. Building on these, we hypothesized roles for PGC-1α in EPC dysfunction. Methods and Results: We used Tie2-Cre-driven PGC-1α conditional null mice (KO), with or without T1DM induction by streptozocine. DM increased PGC-1α mRNA in WT BM-KSL cells whereas this induction was blocked in KO cells. DM decreased the differentiation capacity of WT BM-KSL cells and PB-MNCs, as determined by the formation of primitive (pEPC) and definitive (dEPC) colonies. PGC-1α ablation in KO rescued the defective EPCs differentiation in DM, most notably PB-MNCs formation of dEPC colonies, the known vasculogenic population. Consistent with this, endothelial marker gene expression (KDR, CD31, vWF), EPCs abundance, and EPCs vasculogenic functions of PB-MNCs was blunted by DM but rescued by PGC-1α ablation in KO. Importantly, T2DM induction by high-fat diet feeding also caused reduction in the EPCs abundance and EC gene expression of PB-MNCs, which was rescued by PGC-1α ablation in KO. Conclusions: Our data demonstrate critical new roles for PGC-1α in vasculogenesis. PGC-1α mediates the vasculogenic differentiation defect of circulating EPCs in T1 and T2 DM, in sharp contrast to the anti-migratory effect of EC PGC-1α in mediating diabetic angiostasis. Our data broadens the relevance of PGC-1α in the etiology of diabetic angiopathy, now with a potential to therapeutically restore EPCs vasculogenicity, and may provide an opportunity to improve the efficacy of autologous adoptive EPC therapy for limb and myocardial ischemia in DM.

The effect of bisphosphonates on the endothelial differentiation of mesenchymal stem cells.

The contribution of the local stem cell niche to providing an adequate vascular framework during healing cannot be overemphasized. Bisphosphonates (BPs) are known to have a direct effect on the local vasculature, but their effect on progenitor cell differentiation is unknown. This in vitro study evaluated the effect(s) of various BPs on the differentiation of human placental mesenchymal stem cells (pMSCs) along the endothelial lineage and their subsequent functional and morphogenic capabilities. pMSC multipotency was confirmed by successful differentiation into cells of both the osteogenic and endothelial lineages, as demonstrated by positive Alizarin Red S staining and Ac-LDL uptake. pMSC differentiation in the presence of non-cytotoxic BP concentrations showed that nitrogen containing BPs had a significant inhibitory effect on cell migration and endothelial marker gene expression, as well as compromised endothelial differentiation as demonstrated using von Willebrand factor immunofluorescence staining and tube formation assay. This in vitro study demonstrated that at non-cytotoxic levels, nitrogen-containing BPs inhibit differentiation of pMSCs into cells of an endothelial lineage and affect the downstream functional capability of these cells supporting a multi-modal effect of BPs on angiogenesis as pathogenic mechanism contributing to bone healing disorders such as bisphosphonate related osteonecrosis of the jaws (BRONJ).

Determination of Early and Late Endothelial Progenitor Cells in Peripheral Circulation and Their Clinical Association with Coronary Artery Disease.

The clinical implications of early and late endothelial progenitor cells (EPCs) in coronary artery disease (CAD) remain unclear. We investigated endothelial dysfunction in CAD by simultaneously examining early and late EPC colony formation and gene expression of specific surface markers in EPCs. EPCs were extracted from a total of 83 subjects with (n = 47) and without (n = 36) CAD. Early and late EPC colonies were formed from mononuclear cells extracted from peripheral blood. We found that fewer early EPC colonies were produced in the CAD group (7.2 ± 3.l/well) than those in the control group (12.4 ± 1.4/well, p < 0.05), and more late EPC colonies were produced in the CAD group (0.8 ± 0.2/well) than those in the control group (0.25 ± 0.02/well, p < 0.05). In the CAD group, the relative expression of CD31 and KDR of early and late EPCs was lower than in the control group. These results demonstrate that CAD patients could have increased late EPC density and that early and late EPCs in CAD patients exhibited immature endothelial characteristics. We suggest that changes in EPC colony count and gene expression of endothelial markers may have relation with development of CAD.

Effects of shear stress on endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are adult stem cells that play a central role in neovascularization. EPCs are mobilized from bone marrow into peripheral blood, attach to existing endothelial cells, and then transmigrate across the endothelium into tissues, where they proliferate, differentiate, and form new blood vessels. In the process, EPCs are exposed to shear stress, a biomechanical force generated by flowing blood and tissue fluid flow. When cultured EPCs are exposed to controlled levels of shear stress in a flow-loading device, their bioactivities in terms of proliferation, anti-apoptosis, migration, production of bioactive substances, anti-thrombosis, and tube formation increase markedly. Expression of endothelial marker genes and proteins by EPCs also increases in response to shear stress, and they differentiate into mature endothelial cells. Great advances have been made in elucidating the mechanisms by which mature endothelial cells sense and respond to shear stress, but not in EPCs. Further study of EPC responses to shear stress will be necessary to better understand the physiological and pathophysiological roles of EPCs and to apply EPCs to new therapies in the field of regenerative medicine.

Role of MiR-424 on Angiogenic Potential in Human Dental Pulp Cells

IntroductionGrowing evidence shows microRNAs (miRNAs) regulate numerous cellular processes. The purpose of this study was to investigate whether miRNAs can regulate the commitment of human dental pulp cells (hDPCs) to the angiogenic fate. MethodsThe hDPCs were induced to differentiate into the vascular lineage. Gene expression of endothelial markers (vWF and CD31) on day 7 after induction was analyzed by using quantitative reverse transcription–polymerase chain reaction (qRT-PCR).The miRNA expression profiling of endothelial differentiation was performed by microarray and was validated by qRT-PCR analysis. The hDPCs were infected by recombinant lentivirus to overexpress or knock down miR-424 stably, and the biological effects of miR-424 on the endothelial differentiation of hDPCs were further investigated. The tube formation ability and the amount of endothelial markers (vWF and KDR) were evaluated by Matrigel assay and Western blotting. Target genes of miR-424 were further determined by bioinformatic algorithms and Western blotting. ResultsAfter endothelial differentiation, the expression of vWF and CD31 increased significantly in hDPCs. Microarray data showed that the miR-424 expression level was down-regulated on day 7. The qRT-PCR revealed a time-dependent decrease, with significant differences detected on day 1 and day 7 (P < .05). Knockdown of miR-424 expression in hDPCs promoted endothelial differentiation, with increased tube formation and up-regulated expression of vWF and KDR. In contrast, overexpression of miR-424 inhibited their differentiation. In addition, miR-424 was predicted to target vascular endothelial growth factor and KDR. Overexpression of miR-424 decreased vascular endothelial growth factor and KDR protein levels, whereas miR-424 inhibition significantly elevated them. ConclusionsThis study demonstrated that miR-424 may play a negative role in regulating endothelial differentiation of hDPCs, and inhibition of miR-424 may contribute to dental pulp repair and regeneration.

Abstract 5424: Hypoxia Induces the Histone Demethylase Jmjd3 and Epigenetically Reprograms Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) contribute to vascular repair and postnatal neovascularisation. Cell therapy using EPCs improved neovascularisation and functional recovery after ischemia. However, it has been extensively debated whether EPCs can differentiate into endothelial cells and the mechanisms of EPC differentiation are poorly understood. Endothelial differentiation requires the expression of endothelial marker genes, which are silenced by chromatin dependent repressive mechanisms such as histone H3 lysine 27 trimethylation (H3K27me3) in non-endothelial cells. We observed that induction of hind limb ischemia increased the expression of Jmjd3, which was shown to demethylate H3K27me3 thereby removing repressive histone marks. Therefore, we examined whether EPC might be reprogrammed by hypoxic/ischemic injury through epigenetic pathways. We report here that Jmjd3 was induced when EPCs were exposed to hypoxia for more than 24 hours. Hypoxia additionally stimulated the nuclear translocation of Jmjd3 in EPC as shown by immunostaining (12.0±3.2% in normoxia, 68.5±3.6% in hypoxia, Jmjd3 positive nuclei/whole nuclei). Increased expression and nuclear translocation of Jmjd3 after hypoxic stimulation was associated with a reduction of global H3K27m3. Additionally, chromatin immunoprecipitation showed that the promoter of eNOS and KDR, which is transcriptionally silenced by high levels of H3K27me3 in early EPC under normoxic conditions, is activated by hypoxia. Hypoxia induced a down-regulation of H3K27me3 (0.045±0.017% input in normoxia and 0.024±0.010% input in hypoxia at the eNOS promoter, 0.197±0.067% input in normoxia and 0.126±0.030% input in hypoxia at the KDR promoter) and increased the ratio of active versus repressive chromatin marks at the eNOS and KDR promoter. Consistently, eNOS and KDR mRNA levels in EPC were increased after hypoxia. In summary, these data demonstrate that hypoxia-induced up-regulation and translocation of the histone demethylase Jmjd3 is associated with a reduction of repressive histone marks at the promoter of endothelial genes. These findings indicate that hypoxia may epigenetically modulate endothelial commitment.

Lentiviral vector: a useful tool for transduction of human liver endothelial cells.

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

Lentiviral Transfer of the LacZ Gene into Human Endothelial Cells and Human Bone Marrow Mesenchymal Stem Cells

Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

REVERSIBLY IMMORTALIZED HUMAN ENDOTHELIAL SCAVENGER CELLS TO DEVELOP A BIOARTIFICIAL ORGAN

Purpose: Endothelial cells are responsible for microcirculation and scavenger functions in various organs and seems to be one of the important components for tissue engineering. The present study describes for the first time a strategy of expansion of primary endothelial cell population by transfer of the simian virus 40 large T antigen (SV40T) gene that can be subsequently excised by Cre/loxP site-specific recombination. Methods: To transfer the SV40T gene into human umbilical vein endothelial cells (HUVECs), we constructed a retroviral vector SSR#69 expressing SV40T flanked by a pair of loxP. An adenoviral vector producing Cre recombinase (Ad-Cre) was used for recombination. The gene expression of endothelial markers, uptake of acetylated LDL, and efficacy of Cre/loxP-mediated recombination were evaluated in SV40T-transduced HUVECs. Results: One of SSR#69-transformed HUVECs, HNNT-1, grew economically in culture and showed gene expression of factor VIII, VEGF receptors (flt-1 and KDR/flk-1), CD34, and HGF. The cells demonstrated the uptake-of acetylated LDL. SV40T was efficiently removed following infection of Ad-Cre, while retaining the characteristics of primary HUVECs. Conclusion: We present here for the first time a method to control the expansion of primary human endothelial cells as a source for developing a novel hybrid bioartificial organ.