Transduction Of Endothelial Cells Research Articles

DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway.

7-difluoromethoxy-5,4′-dimethoxy-genistein (DFMG) is a novel active chemical entity, which modulates the function and signal transduction of endothelial cells and macrophages (MPs), and is essential in the prevention of atherosclerosis. In the present study, the activity and molecular mechanism of DFMG on MPs was investigated using a Transwell assay to construct a non-contact co-culture model. Human umbilical vein endothelial cells (HUVE-12), which were incubated with lysophosphatidylcholine (LPC), were seeded in the upper chambers, whereas PMA-induced MPs were grown in the lower chambers. The generation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) were measured using the corresponding assay kits. The proliferation and migration were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound healing assays, respectively. Foam cell formation was examined using oil red O staining and a total cholesterol assay. The protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-κB p65 were detected by western immunoblotting. The secretion of interleukin (IL)-1β was examined using an enzyme-linked immunosorbent assay. It was found that LPC significantly increased the generation of ROS and the release of LDH in HUVE-12 cells. The LPC-injured HUVE-12 cells activated MPs under co-culture conditions and this process was inhibited by DFMG treatment. LPC upregulated the expression levels of TLR4, MyD88 and NF-κB p65, and the secretion of IL-1β in the supernatant of the co-cultured HUVE-12 cells and MPs. These effects were reversed by the application of DFMG. Furthermore, CLI-095 and IL-1Ra suppressed the activation of MPs that was induced by co-culture with injured HUVE-12 cells. These effects were further enhanced by co-treatment with DFMG, and DFMG exhibited synergistic effects with a TLR4-specific inhibitor. Take together, these findings revealed that DFMG attenuated the activation of MP induced by co-culture with LPC-injured HUVE-12 cells. This process was mediated via inhibition of the TLR4/MyD88/NF-κB signaling pathway in HUVE-12 cells.

Endothelial Interferon Regulatory Factor 1 Regulates Lipopolysaccharide-Induced VCAM-1 Expression Independent of NFκB

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB.

A Sema3C Mutant Resistant to Cleavage by Furin (FR-Sema3C) Inhibits Choroidal Neovascularization.

In age-related macular degeneration (AMD), abnormal sub retinal choroidal neovascularization (CNV) is a major cause of blindness. FR-sema3C is a point mutated form of semaphorin-3C that is resistant to cleavage by furin like pro-protein convertases (FPPC). We have found in previous work that FR-sema3C functions as an anti-angiogenic factor. In this study we investigated the possible use of FR-sema3C as an inhibitor of CNV. FR-sema3C inhibits VEGF as well as PDGF-BB signal transduction in endothelial cells and to less extent bFGF induced signal transduction using a mechanism that does not depend upon the binding of VEGF like the drugs that are currently the mainstay treatment for AMD. CNV was induced in eyes of C57 black mice by laser photocoagulation. Intravitreal injection of FR-Sema3C or aflibercept (VEGF-trap) was then used to inhibit CNV formation. Invading choroidal vessels were visualized a week later by injection of FITC-dextran into the circulation, followed by the measurement of the area of the invading blood vessels. Injection of 0.1 μg FR-Sema3C inhibited CNV by 55% (P<0.01) and was as effective as 5 μg aflibercept. FR-sema3C did not display any adverse effects on retinal function following its injection into eyes of healthy mice as assessed by optokinetic reflex (OKR) and Electro-retinogram (ERG) criteria. Furthermore, FR-sema3C did not induce apoptosis in the retina as determined by TUNEL nor was there any discernable structural damage to the retina as assessed by several immuno-histochemical criteria. Our results suggest that FR-sema3C could perhaps be used for the treatment of AMD, and that it may perhaps be of benefit to patients that do not respond well to current treatments relying on VEGF sequestering agents.

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.

Stromal cell-derived factor 2 is critical for Hsp90-dependent eNOS activation.

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.

Plasma Membrane Organization Promotes CD36 Signal Transduction in Endothelial Cells

CD36 is a multiligand scavenger receptor that ligates Thrombospondin-1 (TSP-1) at the surface of endothelial cells and induces their apoptosis. Recent evidences have shown that clustering of CD36 is necessary for signal transduction in macrophages and is regulated by the architecture of the cortical actin cytoskeleton apposed to the plasmalemma. Here, we investigated the role of the cortical actin cytoskeleton and plasma membrane nanodomains in the control of CD36 activation in endothelial cells. Stimulation with multivalent ligands (TSP-1 and anti-CD36 IgM) resulted in the downstream phosphorylation of the Src Family kinase, Fyn. Disruption of the actin cytoskeleton or removal of cholesterol blocked this activation. To gain molecular details on the rearrangement of the receptors during TSP-1 binding, we conducted superresolution approaches (based on PhotoActivated Localization Microscopy or PALM) and quantitative spatial distribution analysis. Endothelial cell lines stably expressing CD36-PAmCherry were generated for that purpose. At steady state, CD36 receptors pre-exist in small clusters (average diameter of 100 nm), in which Fyn was also present. Upon TSP-1 binding, CD36 clusters increased in size (average diameter of 140 nm) and also became denser. The average distance between CD36 molecules in these clusters was in the range of 8 nm compare to 11 nm in the control condition. F-actin depolymerization or cholesterol depletion reduced the capacity of the ligand to induce formation of larger clusters resulting in a decreased recruitment and activation of Fyn. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the organization of CD36 receptors and Fyn before and during TSP-1 stimulation.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

Comparison of viral vectors for gene transfer to corneal endothelial cells

AbstractPurpose Thanks to its anatomical location at the posterior surface of the cornea and its monolayer structure, the corneal endothelium is an ideal target for gene therapy approaches. Lentiviral vectors have been shown by our group to be suitable vectors for the transfer of genes into corneal endothelial cells (EC). Aiming for an alternative to these HIV‐based vectors, it was the goal of this study to determine the suitability of non‐pathogenic adeno‐associated viral vectors (AAV) for gene transfer to EC.Methods Comparison of protein expression after EC transduction using a lentiviral vector or AAV 2/2 with GFP in murine EC (Balb/C) and in human EC (cell line and primary cells)by flow cytometry.Results Following transduction of EC using lentiviral vector, kinetics of the protein expression are considerably different compared to gene transfer using AAV. In contrast to AAV with protein expression showing a plateau after two to three weeks, lentiviral transfer results in a very rapid of reporter protein. Moreover, we detected significant differences in transduction rates between human and murine EC lines as well as betweeen human EC lines and human corneas (plateau at 70% versus 50% GFP‐positive cells with AAV, versus 90‐95% with lentivirus).Conclusion DNA transfer using AAV vectors seems to be an appropriate alternative to lentiviral vectors for gene transfer to EC. Relating to the cultivation of human donor corneas in eye banks over weeks, translation of AAV from bench to bedside, e.g. to reduce apoptosis in corneas, seems to be a promising approach for future gene transfer into donor corneas.

Comparative Study on Signal Transduction in Endothelial Cells After Anti-A/B and Human Leukocyte Antigen Antibody Reaction

Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

Optimization of Magnetic Nanoparticle-Assisted Lentiviral Gene Transfer

Targeting of specific cells and tissues is of great interest for clinical relevant gene- and cell-based therapies. We use magnetic nanoparticles (MNPs) with a ferrimagnetic core (Fe(3)O(4)) with different coatings to optimize MNP-assisted lentiviral gene transfer with focus on different endothelial cell lines. Lentiviral vector (LV)/MNP binding was characterized for various MNPs by different methods (e.g. magnetic responsiveness measurement). Transduced cells were analyzed by flow cytometry, fluorescence microscopy and iron recovery. Cell transduction and cell positioning under physiological flow conditions were performed using different in vitro and ex vivo systems. Analysis of diverse MNPs with different coatings resulted in identification of nanoparticles with improved LV association and enhanced transduction properties of complexes in several endothelial cell lines. The magnetic moments of LV/MNP complexes are high enough to achieve local gene targeting of perfused endothelial cells. Perfusion of a mouse aorta with LV/MNP transduced cells under clinically relevant flow conditions led to local cell attachment at the intima of the vessel. MNP-guided lentiviral transduction of endothelial cells can be significantly enhanced and localized by using optimized MNPs.

Identification of Magnetic Nanoparticles for Combined Positioning and Lentiviral Transduction of Endothelial Cells

The combination of magnetic nanoparticles (MNPs) with a magnetic field is a powerful approach to enable cell positioning and/or local gene therapy. Because physical requirements for MNPs differ between these two applications we have explored whether the use of different MNPs can provide site-specific positioning combined with efficient viral transduction of endothelial cells (ECs). A variety of MNPs was screened for magnetic cell labeling and lentivirus binding. Then two different MNPs were chosen and their combined application was evaluated regarding EC magnetization and transduction efficiency. The combined use of PEI-Mag2 and NDT-Mag1 particles provided both efficient lentiviral transduction and high magnetic responsiveness of ECs that could be even retained within the vascular wall under flow conditions. The use of these MNPs did not affect biological characteristics of ECs like surface marker expression and vascular network formation. Importantly, with this method we could achieve an efficient functional overexpression of endothelial nitric oxide synthase in ECs. The application of two different MNPs provides optimal results for magnetic labeling of ECs in combination with viral transduction. This novel approach could be very useful for targeted gene therapy ex vivo and site-specific cell replacement in the vascular system.

Optimizing Adenoviral Transduction of Endothelial Cells under Flow Conditions

To target adenoviral vectors to cells of the vasculature and shielding vectors from inactivation by the immune system. Complexes of reporter gene expressing adenoviral vectors with positively charged magnetic nanoparticles were formed by electrostatic interaction in presence or absence of additional negatively charged poly(ethylene glycol)-based polymer. Transduction of HUVEC was analyzed in vitro under flow. Protection from inactivation by the immune system was analyzed by pre-incubation of AdV and complexes with neutralizing antibodies and subsequent reporter protein analysis of infected cells. Physical association of AdV with MNP and polymers was demonstrated by radioactive labelling of components and co-sedimentation in a magnetic field. Ad-MNP+/-polymer resulted in efficient transduction of HUVEC, depending on MOI and flow rate in presence of magnetic field, whereas no transduction was observed without complex formation with MNP or in absence of magnetic field. Association with MNP did result in protection from neutralizing antibodies, with slightly increased protection provided by the polymer. Complex formation of AdV with MNP is a viable means for targeting of vectors to areas of magnetic field gradient. Additional coating with polymer might proof useful in protection from inactivation by the immune system.

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

BackgroundADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis ‘senses’ NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs.Methodology/Principal FindingsUsing biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47phox subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS.Conclusion/SignificanceThese results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can ‘sense’ redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.

42-OR: HLA class II antibodies elicit intracellular signal transduction in endothelial cells

42-OR: HLA class II antibodies elicit intracellular signal transduction in endothelial cells

Viral vectors for gene transfer into corneal endothelial cells

AbstractPurpose Corneal endothelium is an ideal target fot gene therapy approached thanks to its anatomical location at the posterior surface of the cornea and its monolayer character. Lentiviral vectors have been shown by our group to be suitable vectors for the transfer of DNA into corneal endothelial cells (EC). Searching for an alternative to these HIV‐based vectors, aim of this study was to determine the suitability of non‐pathogenic adeno‐associated viral vectors (AAV) for gene transfer to EC.Methods Flowcytometric comparison of protein expression after transduction of EC using a lentiviral vector or AAV 2/2 with GFP in murine EC (Balb/C) and in human EC (cell line and primary cells). Proof of principle experiment to demonstrate the functionality of lentiviral gene transfer of the anti‐apoptotic proteins Bcl‐xL.Results Kinetics of the protein expression after transduction of EC using lentiviral vector are considerably different compared to gene transfer using AAV. Contrary to AAV overexpression of the reporter protein after lentiviral gene transfer occurs very rapid. Moreover, we detected significant differences in transduction rates between human and murine EC lines as well as betweeen human EC lines and human corneas.Conclusion AAV vectors seem to be an alternative to lentiviral vectors for gene tranfer to EC. Considering the storage of human donor corneas in eye banks in organ culture over four weeks, translation of AAV from bech to bedside, e.g. to reduce apoptosis in corneas, seems to be feasible.

Virale Vektoren für den Gentransfer in korneale Endothelzellen

Corneal endothelium is an interesting target for in vitro gene transfer strategies as it is readily accessible thanks to its anatomic structure as a monolayer and its direct contact to culture medium. Whereas the use of adenoviruses as viral vectors (carriers) to endothelial cells (EC) has been described as problematic as to its immunogenicity, lentiviruses and adeno-associated viruses (AAV) are potent vectors for the transfer of genetic DNA into EC. Lentiviral vectors, developed on the basis of HI-viruses, can integrate the transferred gene into the host DNA and thus lead to a permanent protein expression. Evaluating apathogen alternatives to lentiviral vectors for humans, we herein compared non-integrating AAV to lentiviral gene transfer. A comparison was made of the kinetics of expression of a green fluorescent protein after transduction using a lentiviral vector and AAV 2 / 2 in a murine EC line, human EC line and human primary cells (flow cytometry). A proof of principle experiment was conducted to demonstrate the function after lentiviral gene transfer of the anti-apoptotic gene Bcl-xL. The kinetics of protein expression after transduction of EC using a lentiviral or an AAV vector show fundamental differences. Contrary to gene transfer using AAV, a high expression of the reporter protein was readily detectable only hours after transduction using the lentiral vector. In addition, we could demonstrate distinct differences in protein expression characteristics between human and murine EC as well as human EC line and primary human EC. Function could be demonstrated by showing a significant reduction in apoptosis in both murine and human EC. AAV vectors are an alternative to lentiviral vectors for gene transfer to corneal EC. Given a cultivation time of donor corneas of up to 4 weeks before transplantation, translation to eye banking, e. g., to decrease apoptosis in corneal allografts, is conceivable.

Genome-Wide Approaches Reveal Functional Interleukin-4-Inducible STAT6 Binding to the Vascular Cell Adhesion Molecule 1 Promoter

Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.

Radially symmetric endothelial cell replacement and lentiviral targeting in vessels by the use of magnetic nanoparticles (MNPs)

Endothelial cell damage often occurs in vascular disease such as atherosclerosis resulting in loss of vasoregulation and/or thrombosis. To restore endothelial function we have established novel methods to either combine site-specific endothelial cell positioning with lentiviral transduction in arteries or directly transduce the vascular endothelium in situ. Therefore, we have designed a special magnetic device to obtain a radially symmetric endothelial cell or lentivirus deposition on the vascular wall. Lentivirus is coupled to magnetic nanoparticles (MNPs) enabling the efficient transduction of endothelial cells by application of magnetic force. As a proof of concept we show that bovine pulmonary arterial endothelial cells (bPAECs) and human umbilical vein endothelial cells (HUVECs) are efficiently transduced with complexes of MNPs and lentiviral vectors carrying a GFP expression cassette. These cells can be positioned in a radially symmetric manner in an ex vivo flow-loop system. Moreover, this technique also allows for the direct lentiviral transduction of the murine endothelium in situ, which is proven by GFP expression in the vessel after 4–6 days in culture. We plan to use this approach in combination with the application of therapeutic genes e.g. the endothelial nitric oxide synthase (eNOS) to restore vascular endothelial function ex vivo and in vivo.