Departments of 1Pharmacology, 2Urology, and 3Structural and Cellular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112
Submitted 9 April 2003 ; accepted in final form 9 July 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
adenoviral vector; nitric oxide; gene expression; differentiation; gene therapy
Gene-modified ex vivo expanded adult stem cells are attractive for use in gene therapy, because they avoid some of the risks and disadvantages associated with direct in vivo delivery of viral vectors, nonviral vectors, or gene-modified ex vivo expanded differentiated cells (40). Marrow stromal cells (MSCs), also known as mesenchymal stem cells, are nonhematopoietic adult stem cells from bone marrow, are relatively easy to isolate and expand ex vivo, and have multidifferentiation potential (10, 17, 18, 21, 24, 25, 33, 38, 39, 41, 45). Therefore, MSCs may be a useful vehicle for gene delivery in adult stem cell-based gene therapy (2, 26, 47). For ex vivo gene transfer, adenoviral vectors have several advantages over other vectors, such as their ability to infect quiescent cells, a broad host range, the ease of preparation of high-titer viral stock, and high-level transgene expression (9). The objective of this study was to determine whether ex vivo expanded rat MSCs (rMSCs) can be transduced with adenovirus containing eNOS and to ascertain whether the cells retain multipotential differentiation capability after adenoviral-mediated eNOS gene transfer.
In this study, we present data demonstrating that adenoviral vectors can be used to gene engineer ex vivo expanded rMSCs and that high-level eNOS expression can be achieved, underscoring the potential utility of this novel method for adult stem cell-based gene therapy.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and ex vivo expansion of rMSCs. rMSCs were isolated as previously described (7, 30, 35, 43, 44). Briefly, 6-wk-old male Brown Norway rats (Harlan, San Diego, CA) were euthanized with CO2. Under sterile conditions, femurs and tibias were removed and placed in the culture medium for rMSCs [-MEM (GIBCO Invitrogen, Grand Island, NY), 20% fetal bovine serum (GIBCO Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 ng/ml amphotericin B (Atlanta Biologicals, Norcross, GA), and 2 mM L-glutamine (GIBCO Invitrogen)]. Both ends of femurs and tibias were removed, and the bone marrow was flushed out using a 21-gauge needle attached to a 10-ml syringe filled with the culture medium for rMSCs. The bone marrow cells were filtered through a cell strainer with 70-µm nylon mesh (BD Bioscience, Bedford, MA), and the cells from one rat were plated in a 75-cm2 tissue culture flask (BD Bioscience). The cells were incubated in the culture medium for rMSCs at 37°C with 5% humidified CO2, and rMSCs were isolated by their adherence to tissue culture plastic. Fresh culture medium was added and replaced to remove nonadherent cells every 2-3 days. The adherent rMSCs were grown to confluency (
7 days, defined as rMSCs at passage 0), harvested with 0.25% trypsin and 1 mM EDTA for 5 min at 37°C, diluted 1:3, replated in culture flasks, and again grown to confluency for further expansion. rMSCs at passage 1-5 were used for all experiments.
Transduction with adenoviral vectors. rMSCs were plated at a density of 10,000 cells/cm2 in six-well plates or T-25 flasks (BD Bioscience) and cultured overnight. The cells were then exposed to fresh culture medium containing adenoviral vector at various multiplicities of infection (MOI, defined as pfu/cell) for 48 h. Three separate experiments were carried out, each in triplicate. Cell viability was determined using the trypan blue exclusion method.
Immunohistochemistry. Immunohistochemical analysis for eNOS transgene expression in culture was carried out as described previously (22). Briefly, cells in six-well plates were rinsed with PBS, fixed with cold methanol (-20°C) for 5 min, and washed with PBS twice, and the immunostaining was performed using the mouse anti-eNOS monoclonal antibody (BD Transduction Laboratories, San Diego, CA; 1:100 dilution) and the mouse Immunocruz staining system (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were then counterstained with hematoxylin (Rowley Biochemical, Danvers, MA), and expression of eNOS transgene in rMSCs was evaluated in situ by light microscopy scoring of cells expressing eNOS. The eNOS-positive brown cells found in two to three microscopic fields (x25) were counted and expressed as a percentage of the total number of cells in those fields. Immunohistochemical analysis of eNOS-positive Ad5RSVeNOS-transduced rMSCs in rat corpus cavernosum was carried out as described above, except 5-µm penile cross sections on glass slides were rinsed with PBS twice before being immunostained. Penile sections were then counterstained with hematoxylin (Rowley Biochemical), mounted with VectaMount mounting medium (Vector Laboratories, Burlingame, CA), and covered with coverslips, and the localization of eNOS-positive Ad5RSVeNOS-transduced rMSCs in rat corpus cavernosum was analyzed by light microscopy.
X-gal histochemistry for -galactosidase activity. Cells in six-well plates were rinsed with PBS, fixed in a PBS solution containing 2% formaldehyde and 0.2% glutaraldehyde (Sigma, St. Louis, MO) for 5 min, washed with PBS twice, and incubated in the X-gal staining solution [5 mM K ferricyanide, 5 mM K ferrocyanide, 2 mM MgCl2, and 1 mg/ml X-gal (Sigma), prepared in PBS] overnight at 37°C in darkness. Cells were then rinsed with PBS, and expression of ntlacZ transgene in rMSCs was evaluated by light microscopy scoring of cells expressing nuclear-targeted
-galactosidase activity. The
-galactosidase-positive blue cells found in three microscopic fields (x25) were counted and expressed as a percentage of the total number of cells in those fields.
Western blot analysis. Western blot analysis for eNOS transgene expression in culture was carried out using the whole cell lysate. Briefly, cells in six-well plates or T-25 flasks were rinsed with cold PBS, drained, lysed with a buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml leupeptin, and 574 µM phenylmethylsulfonyl fluoride (Sigma; prepared in PBS), and scraped into a 1.5-ml centrifuge tube. The sample was incubated for 45 min on ice and centrifuged at 15,000 g for 20 min, and the supernatant was collected. Protein content of the whole cell lysate was quantified colorimetrically using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Whole cell lysate (5 µg) was loaded onto a 4-20% Tris-glycine gel (ICN Biomedicals, Aurora, OH). After electrophoresis, the protein was transferred onto a nitrocellulose membrane by electroelution. Immunodetection was performed with mouse anti-eNOS monoclonal antibody (BD Transduction Laboratories; 1:2,500 dilution) and rabbit anti--tubulin polyclonal antibody (Santa Cruz Biotechnology; 1:2,500 dilution). The secondary antibodies were horseradish peroxidase conjugated to goat anti-mouse IgG (Santa Cruz Biotechnology; 1:4,000 dilution) or anti-rabbit IgG (Santa Cruz Biotechnology; 1:4,000 dilution). The nitrocellulose membrane was processed using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Human aortic endothelial cell lysate (5 µg; BD Transduction Laboratories) was used as a positive control. Western blot analysis for eNOS transgene expression in rat cavernosal tissue was carried out as described above, except 60 µg of penile homogenate per lane were loaded.
In vitro differentiation of rMSCs into osteoblasts or adipocytes. In vitro differentiation of rMSCs into the osteoblast or adipocyte lineage was carried out as previously described (7, 35, 43). Briefly, cells in six-well plates were treated with culture medium for rMSCs plus osteogenic supplement [1 x 10-5 mM dexamethasone, 0.2 mM ascorbic acid, and 10 mM -glycerol phosphate (Sigma)] or adipogenic supplement [0.5 µM hydrocortisone, 500 µM isobutylmethylxanthine, and 60 µM indomethacin (Sigma)]. The differentiation medium was changed every 3 days until day 21. To assess mineral deposition, cells were rinsed with PBS, fixed in cold methanol (-20°C) for 10 min, briefly washed twice with distilled H2O (dH2O), stained with 2% alizarin red S (pH 4.1; Sigma) for 15 min, and washed five times with dH2O. To assess lipid droplet formation, cells were rinsed with PBS, fixed in 10% formalin (Sigma) for 1 h, briefly washed twice with dH2O, stained with a freshly prepared oil red O solution for 15 min, and washed with dH2O. The oil red O solution was prepared by mixing three parts of an oil red O stock solution [0.5% in isopropanol (Sigma)] with two parts of dH2O and filtering it through a 0.45-µm-pore filter.
Intracavernosal injection of Ad5RSVeNOS-transduced rMSCs into aged rats and histology. rMSCs were transduced with Ad5RSVeNOS at MOI 300 for 48 h. The virus-containing culture medium was removed, and the cells were washed three times with PBS. These Ad5RSVeNOS-transduced rMSCs were then harvested with trypsin-EDTA and washed with PBS, and a cell suspension at a concentration of 12,500 cells/µl was prepared in PBS. The cell suspension was put on ice until the rats were anesthetized. All in vivo experiments were performed on 25-mo-old male Brown Norway rats in accordance with institutional and National Institutes of Health guidelines for the care and use of laboratory animals. These aged rats were anesthetized with pentobarbital sodium (30 mg/kg, ip; Sigma) and placed in a supine position on a heated surgical table. Under sterile conditions, the penis was exposed and 40 µl of cell suspension or 40 µl of PBS were injected into the corpus cavernosum with a syringe and a 25-gauge needle. A total of 500,000 cells or 40 µl of PBS was injected into each rat, and six rats per group were used in the experiment. Immediately before injection, blood drainage via the dorsal vein was halted by circumferential compression of the penis at the base with an elastic band. The compression was released 1 min after the injection. The rat was monitored until recovery from the anesthetic and then returned to the animal care facility. At 7 days after intracavernosal injection of Ad5RSVeNOS-transduced rMSCs or PBS, rats were deeply anesthetized with pentobarbital sodium (80 mg/kg, ip; Sigma) and intracardiacally perfused with 200 ml of ice-cold PBS. For immunohistochemistry, the penis was removed, cut into 5-mm sagittal fragments, and fixed in ice-cold 4% paraformaldehyde in PBS (US Biochemicals, Cleveland, OH) at 4°C overnight. The penis was then transferred to 30% sucrose in PBS (Sigma) in a 50-ml conical tube at 4°C overnight. The tissue was embedded in OCT compound (Triangle Biomedical Sciences, Durham, NC), snap frozen in liquid nitrogen, and stored at -70°C. Penile cross sections (5 µm) were then prepared with a cryostat and mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). To prepare homogenates for Western blot analysis, the penis was excised, quickly frozen in liquid nitrogen, and stored at -70°C. Cavernosal tissue was then homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in an ice-cold buffer (5 mM HEPES, pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 300 mM NaCl), incubated on ice for 30 min, and centrifuged twice at 15,000 g at 4°C for 20 min, and the supernatant was collected. Protein content of the homogenate was quantified colorimetrically using the bicinchoninic acid protein assay (Pierce).
Statistical analysis. Values are means ± SE and were analyzed statistically using a t-test or a one-way analysis of variance followed by post hoc analysis with Tukey's test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Western blot analysis for eNOS. eNOS expression was assessed in Ad5RSVeNOS-transduced rMSCs, Ad5RSVntlacZ-transduced rMSCs, and control rMSCs using Western blot analysis. No constitutive eNOS expression was detected in control rMSCs or Ad5RSVntlacZ-transduced rMSCs. Expression of eNOS was detected in Ad5RSVeNOS-transduced rMSCs, and dose-dependent eNOS transgene expression was also observed. Furthermore, eNOS expression levels are much higher in Ad5RSVeNOS-transduced rMSCs than in endothelial cells (Fig. 1B).
Differentiation of Ad5RSVeNOS-transduced rMSCs in vitro. To investigate whether rMSCs retain multidifferentiation potential after adenoviral-mediated eNOS gene transfer, rMSCs were transduced with Ad5RSVeNOS at MOI 2,000 for 48 h and further grown in the presence of differentiation media. After exposure to osteogenic medium, mineral deposition was observed (Fig. 3, A-D). Moreover, after exposure to adipogenic medium, the cells exhibited lipid droplets. Thus the osteogenic and adipogenic potential of Ad5RSVeNOS-transduced rMSCs was retained. The percentage of differentiated cells in control and Ad5RSVeNOS-transduced rMSCs was counted. The percentage of differentiated osteoblasts in control and Ad5RSVeNOS-transduced rMSCs was 59 ± 2% and 59 ± 3% (mean ± SE, n = 3, P > 0.05 by t-test), respectively. The percentage of differentiated adipocytes in control and Ad5RSVeNOS-transduced rMSCs was 37 ± 1% and 35 ± 2% (n = 3, P > 0.05 by t-test), respectively. Therefore, these data indicate that there is no statistical difference between the differentiation potential of control and Ad5RSVeNOS-transduced rMSCs in osteogenic or adipogenic medium.
|
The expression of eNOS in these differentiated cells was also assessed. The percentage of eNOS-positive cells at day 21 in osteogenic medium-treated cells was 9 ± 3% (mean ± SE, n = 3). The percentage of eNOS-positive cells at day 21 in adipogenic medium-treated cells was 33 ± 2% (n = 3). The differentiated osteoblasts and adipocytes are still positive for eNOS (Fig. 3, E and F).
Persistence of adenoviral-mediated eNOS transgene expression in vitro. To study the persistence of eNOS transgene expression in vitro, rMSCs were transduced with Ad5RSVeNOS at MOI 300 for 48 h. The virus-containing culture medium was then removed. The cells were washed three times with PBS and further incubated in low-serum medium (-MEM with 2% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 25 ng/ml amphotericin B, and 2 mM L-glutamine) for 21 days. The low-serum medium was changed every 2-3 days, and eNOS expression was assessed at various times after transduction. The number of cells expressing eNOS was >60% at day 2 and >30% at day 21 (Fig. 4).
|
Intracavernosal injection of Ad5RSVeNOS-transduced rMSCs. The purpose of this in vivo study was to assess the capability of Ad5RSVeNOS-transduced rMSCs to express eNOS in the rat corpus cavernosum. For these experiments, 5 x 105 Ad5RSVeNOS-transduced rMSCs (MOI 300) were injected into the corpus cavernosum of aged Brown Norway rats. At 7 days after intracavernosal injection, significant numbers of the transplanted eNOS-positive Ad5RSVeNOS-transduced rMSCs were identified within the proximal, mid, and distal corporal sinusoids. Immunohistochemical localization of eNOS was qualitatively more abundant in the endothelium of the rats transplanted with the rMSCs expressing eNOS (Fig. 5). Western blot analysis further demonstrated that the expression of eNOS protein was increased in the cavernosal tissue 7 days after intracavernosal injection of Ad5RSVeNOS-transduced rMSCs (Fig. 5). To determine whether overexpression of cavernosal eNOS protein would influence erectile function in aged rats in vivo, intracavernosal pressure changes in response to cavernosal nerve stimulation were measured as previously described (4-6, 14). The changes in intracavernosal pressure were determined 7 days after treatment with PBS, Ad5RSVntlacZ-transduced rMSCs (MOI 300), and Ad5RSVeNOS-transduced rMSCs (MOI 300). The increase in intracavernosal pressure and total area under the erectile curve (cmH2O·s, AUC) in response to cavernosal nerve stimulation (5 V) were significantly greater in the Ad5RSVeNOS-transduced rMSC-treated aged rats (73.4 ± 6.4 cmH2O and 6,078 ± 289 AUC, n = 6; Fig. 5) than in the PBS-treated aged rats (48.8 ± 5.1 cmH2O and 3,026 ± 155 AUC, P < 0.05, n = 6) or the Ad5RSVntlacZ-transduced rMSC-treated rats (46.2 ± 4.8 cmH2O and 2,861 ± 329 AUC, P < 0.05, n = 5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was previously reported that ex vivo expanded human MSCs (hMSCs) can be transduced with adenoviral vector containing the reporter gene lacZ (19, 46). However, the data are not consistent. In one study, only a small population of hMSCs (20%) were transduced with the adenoviral vector. Neither the MOI (range 250-2,000) nor the period of time the cells were exposed to the virus (>6 h) caused a variation in the percentage of transduced cells (19). In the other study, adenoviral-mediated gene transfer to hMSCs was shown to be dose dependent. The transduction efficiency at MOI 1,000-1,500 was >90%, and the RSV promoter was more active than the cytomegalovirus promoter in expressing the lacZ gene in hMSCs (46). In the present experiments, we demonstrate that adenoviral-mediated gene transfer to rMSCs is dose dependent and that the RSV promoter can be used to express the reporter gene ntlacZ and the therapeutic gene eNOS.
For gene therapy in vascular diseases such as erectile dysfunction and pulmonary hypertension, in vivo and ex vivo strategies have been used to deliver therapeutic gene products such as NO and calcitonin gene-related peptide (CGRP) into the corpus cavernosum or the lung. In vivo gene therapy strategy includes direct transfer of adenovirus encoding eNOS or CGRP, which was mainly carried out in our laboratory. Previous studies demonstrate that direct in vivo injection of adenovirus containing eNOS or prepro-CGRP attenuated pulmonary hypertension and augmented erectile responses in the rat and mouse (4-6, 13, 14). However, the disadvantage of this strategy includes a local inflammatory response and the random expression of the transgene in almost all cell types.
An ex vivo gene therapy approach is direct injection of eNOS- or CGRP gene-modified carrier cells into an organ. The therapeutic gene-modified carrier cells are used as vehicles to locally deliver therapeutic gene products such as NO and CGRP. The major advantage of this strategy is that the carrier cells can be manipulated ex vivo. However, the criteria for choosing carrier cells are very strict and require that the cells can be easily isolated and ex vivo expanded. It is also important that the cells efficiently express transgenes such as eNOS and CGRP and that the cells survive for a long period of time in vivo and do not elicit a strong immune response. In previous studies, only endothelial cells have been employed in cell transplantation experiments (48). In this previous study, as a first step toward a cell-based gene therapy for erectile dysfunction, autologous microvessel endothelial cells were transplanted into the rat corpus cavernosum. After 2-15 days, the transplanted endothelial cells adhered and persisted in corporal sinusoids. However, another study demonstrates that adenoviral-mediated gene transfer of eNOS to human endothelial cells inhibits endothelial cell proliferation (49). A major advantage of using endothelial cells is that they can be used for autologous transplantation. However, inasmuch as endothelial cells are difficult to obtain from human patients and difficult to manipulate in culture, the use of these cells is limited. Furthermore, transplantation of allogeneic endothelial cells from human donor or human fetus will elicit host immune rejection and can raise ethical issues.
To explore an alternative approach, we propose the use of MSCs as carriers for ex vivo gene therapy for erectile dysfunction and pulmonary hypertension, because these cells fulfill the criteria for carrier cells. First, hMSCs can be easily obtained from the bone marrow of human patients by adherence to cell culture plastic, readily ex vivo expanded, and efficiently gene engineered (10, 15, 16, 18, 19, 28, 38, 43, 46). Therefore, gene-modified ex vivo expanded MSCs can be used as carrier cells to locally deliver a therapeutic gene product after autologous transplantation. In an intracerebral infusion study, rMSCs were transduced with retroviruses encoding tyrosine hydroxylase and GTP cyclohydrolase I. The cells synthesized L-DOPA in vitro and retained their multipotentiality after retroviral transduction. The cells were injected into the striatum of 6-hydroxydopamine-lesioned rats, and the transgene expression lasted for 9 days. There was a significant reduction in apomorphine-induced head rotation in the rat, and the cells engrafted and survived for 87 days (43). Second, MSCs do not elicit a host immune rejection response and can survive for a long period of time in vivo after autologous transplantation (17, 25, 38, 41). Finally, MSCs are capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes, and other cell types (21, 24, 33, 39, 45). Therefore, these cells can be used in adult stem cell-based therapy for the regeneration of mesenchymal tissues such as bone, cartilage, fat, and muscle. In one study, hMSCs were transplanted in utero into the sheep fetus. The cells engrafted in the fetus and persisted in multiple tissues in the neonate for as long as 13 mo and differentiated into chondrocytes, adipocytes, myocytes, cardiomyocytes, bone marrow stromal cells, and thymic stroma (33). In another study, swine MSCs were implanted into the infarct area of a swine myocardial infarct model. Microscopic and sonomicrometry analysis demonstrated that implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improved cardiac function (45). Therefore, eNOS- or CGRP-modified MSCs should be able to locally deliver therapeutic gene products such as NO and CGRP after autologous transplantation, and the cells may also be able to replace injured host cells after in vivo differentiation under the influence of local tissue microenvironment.
To combine gene therapy and adult stem cell procedures, we are pursuing the potential of developing an improved therapy for erectile dysfunction and pulmonary hypertensive disorders using MSCs transduced with therapeutic genes such as eNOS and prepro-CGRP. Our initial in vivo study in aged rats indicates that intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum. We have shown that injection of adenoviral vectors coding for eNOS and CGRP improves erectile function, and from our preliminary experiments with Ad5RSVeNOS-transduced rMSCs, we hypothesize that injection of eNOS-transduced rMSCs will improve erectile function and may have the advantage of causing less inflammation (3-6, 13, 14). Future erectile function time course studies and an evaluation of cavernosal nerve-mediated and agonist-induced erectile responses will be undertaken to determine whether eNOS-expressing rMSCs improve erectile function over long periods of time in aged rats.
In summary, the results of the present study show for the first time the successful adenoviral gene transfer of eNOS to ex vivo expanded rMSCs. The Ad5RSVeNOS-transduced rMSCs produced high levels of eNOS expression that persisted in culture for >21 days. The cells retained their multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Moreover, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum and improved erectile function in aged rats. These data suggest that this novel adult stem cell-based gene therapy strategy may represent a new form of therapy for the treatment of disorders in which eNOS activity is reduced.
![]() |
DISCLOSURES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bianco P, Riminucci M, Gronthos S, and Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19: 180-192, 2001.
3. Bivalacqua TJ, Armstrong JS, Biggerstaff J, Abdel-Mageed AB, Kadowitz PJ, Hellstrom WJ, and Champion HC. Gene transfer of extracellular SOD to the penis reduces O2 -· and improves erectile function in aged rats. Am J Physiol Heart Circ Physiol 284: H1408-H1421, 2003.
4. Bivalacqua TJ, Champion HC, Abdel-Mageed AB, Kadowitz PJ, and Hellstrom WJ. Gene transfer of prepro-calcitonin gene-related peptide restores erectile function in the aged rat. Biol Reprod 65: 1371-1377, 2001.
5. Bivalacqua TJ, Champion HC, Mehta YS, Abdel-Mageed AB, Sikka SC, Ignarro LJ, Kadowitz PJ, and Hellstrom WJ. Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat. Int J Impot Res 12 Suppl 3: S8-S17, 2000.[ISI][Medline]
6. Bivalacqua TJ, Usta MF, Champion HC, Adams D, Namara DB, Abdel-Mageed AB, Kadowitz PJ, and Hellstrom WJ. Gene transfer of endothelial nitric oxide synthase partially restores nitric oxide synthesis and erectile function in streptozotocin diabetic rats. J Urol 169: 1911-1917, 2003.[ISI][Medline]
7. Bohic S, Pilet P, and Heymann D. Effects of leukemia inhibitory factor and oncostatin M on bone mineral formed in in vitro rat bone-marrow stromal cell culture: physicochemical aspects. Biochem Biophys Res Commun 253: 506-513, 1998.[ISI][Medline]
8. Bonnerot C, Rocancourt D, Briand P, Grimber G, and Nicolas JF. A -galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc Natl Acad Sci USA 84: 6795-6799, 1987.[Abstract]
9. Brenner M. Gene transfer by adenovectors. Blood 94: 3965-3967, 1999.
10. Bruder SP, Jaiswal N, and Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 64: 278-294, 1997.[ISI][Medline]
11. Burnett AL, Lowenstein CJ, Bredt DS, Chang TS, and Snyder SH. Nitric oxide: a physiologic mediator of penile erection. Science 257: 401-403, 1992.[ISI][Medline]
12. Cartledge J, Minhas S, and Eardley I. The role of nitric oxide in penile erection. Expert Opin Pharmacother 2: 95-107, 2001.[Medline]
13. Champion HC, Bivalacqua TJ, D'Souza FM, Ortiz LA, Jeter JR, Toyoda K, Heistad DD, Hyman AL, and Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the lung of the mouse in vivo. Effect on agonist-induced and flow-mediated vascular responses. Circ Res 84: 1422-1432, 1999.
14. Champion HC, Bivalacqua TJ, Hyman AL, Ignarro LJ, Hellstrom WJ, and Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. Proc Natl Acad Sci USA 96: 11648-11652, 1999.
15. Cherington V, Chiang GG, McGrath CA, Gaffney A, Galanopoulos T, Merrill W, Bizinkauskas CB, Hansen M, Sobolewski J, Levine PH, Greenberger JS, and Hurwitz DR. Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion. Hum Gene Ther 9: 1397-1407, 1998.[ISI][Medline]
16. Chuah MK, Van Damme A, Zwinnen H, Goovaerts I, Vanslembrouck V, Collen D, and Vandendriessche T. Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum Gene Ther 11: 729-738, 2000.[ISI][Medline]
17. Clark BR and Keating A. Biology of bone marrow stroma. Ann NY Acad Sci 770: 70-78, 1995.[Abstract]
18. Colter DC, Class R, DiGirolamo CM, and Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97: 3213-3218, 2000.
19. Conget PA and Minguell JJ. Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells. Exp Hematol 28: 382-390, 2000.[ISI][Medline]
20. Davidson BL, Doran SE, Shewach DS, Latta JM, Hartman JW, and Roessler BJ. Expression of Escherichia coli -galactosidase and rat HPRT in the CNS of Macaca mulatta following adenoviral-mediated gene transfer. Exp Neurol 125: 258-267, 1994.[ISI][Medline]
21. Deng W, Obrocka M, Fischer I, and Prockop DJ. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun 282: 148-152, 2001.[ISI][Medline]
22. D'Souza FM, Sparks RL, Chen H, Kadowitz PJ, and Jeter JR Jr. Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation. Am J Physiol Cell Physiol 284: C191-C199, 2003.
23. Edwards JC, Ignarro LJ, Hyman AL, and Kadowitz PJ. Relaxation of intrapulmonary artery and vein by nitrogen oxide-containing vasodilators and cyclic GMP. J Pharmacol Exp Ther 228: 33-42, 1984.[Abstract]
24. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, and Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279: 1528-1530, 1998.
25. Friedenstein AJ, Chailakhyan RK, and Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20: 263-272, 1987.[ISI][Medline]
26. Gronthos S and Simmons PJ. The biology and application of human bone marrow stromal cell precursors. J Hematother 5: 15-23, 1996.[Medline]
27. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, and Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377: 239-242, 1995.[ISI][Medline]
28. Hurwitz DR, Kirchgesser M, Merrill W, Galanopoulos T, McGrath CA, Emami S, Hansen M, Cherington V, Appel JM, Bizinkauskas CB, Brackmann HH, Levine PH, and Greenberger JS. Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells. Hum Gene Ther 8: 137-156, 1997.[ISI][Medline]
29. Ignarro LJ, Burke TM, Wood KS, Wolin MS, and Kadowitz PJ. Association between cyclic GMP accumulation and acetylcholine-elicited relaxation of bovine intrapulmonary artery. J Pharmacol Exp Ther 228: 682-690, 1984.[Abstract]
30. Javazon EH, Colter DC, Schwarz EJ, and Prockop DJ. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells 19: 219-225, 2001.
31. Knorr D. Serious Event on NIH Human Gene Transfer Protocol 9512-139. A Phase I Study of Adenovector-Mediated Gene Transfer to Liver in Adults With Partial Ornithine Transcarbamylase Deficiency. Bethesda, MD: memorandum, National Institutes of Health, Office of Recombinant DNA Activities, 21 September 1999.
32. Leclercq B, Jaimes EA, and Raij L. Nitric oxide synthase and hypertension. Curr Opin Nephrol Hypertens 11: 185-189, 2002.[ISI][Medline]
33. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM, Deans R, Marshak DR, and Flake AW. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6: 1282-1286, 2000.[ISI][Medline]
34. Lozier JN, Metzger ME, Donahue RE, and Morgan RA. Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood 94: 3968-3975, 1999.
35. Malaval L, Modrowski D, Gupta AK, and Aubin JE. Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J Cell Physiol 158: 555-572, 1994.[ISI][Medline]
36. Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, and Murphy TJ. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 90: 2092-2096, 1992.[ISI][Medline]
37. Ooboshi H, Chu Y, Rios CD, Faraci FM, Davidson BL, and Heistad DD. Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol 273: H265-H270, 1997.
38. Owen M and Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 136: 42-60, 1988.[ISI][Medline]
39. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, and Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143-147, 1999.
40. Poulsom R, Alison MR, Forbes SJ, and Wright NA. Adult stem cell plasticity. J Pathol 197: 441-456, 2002.[ISI][Medline]
41. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276: 71-74, 1997.
42. Schmidt HH and Walter U. NO at work. Cell 78: 919-925, 1994.[ISI][Medline]
43. Schwarz EJ, Alexander GM, Prockop DJ, and Azizi SA. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 10: 2539-2549, 1999.[ISI][Medline]
44. Schwarz EJ, Reger RL, Alexander GM, Class R, Azizi SA, and Prockop DJ. Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro. Gene Ther 8: 1214-1223, 2001.[ISI][Medline]
45. Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, and Martin BJ. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 73: 1919-1925, 2002.
46. Toma C, Pittenger MF, Cahill KS, Byrne BJ, and Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105: 93-98, 2002.
47. Van Damme A, Vanden Driessche T, Collen D, and Chuah MK. Bone marrow stromal cells as targets for gene therapy. Curr Gene Ther 2: 195-209, 2002.[Medline]
48. Wessells H and Williams SK. Endothelial cell transplantation into the corpus cavernosum: moving towards cell-based gene therapy. J Urol 162: 2162-2164, 1999.[ISI][Medline]
49. Zanetti M, Katusic ZS, and O'Brien T. Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells. J Vasc Res 37: 449-456, 2000.[ISI][Medline]