Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer͞promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 g͞ml of canine factor IX was detected in the plasma of mice injected with 5.6 ؋ 10 11 particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunof luorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adenoassociated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.Hemophilia B is an X-linked bleeding disorder resulting from a defect or deficiency in blood coagulation factor IX. Recently, three hemophilia B knockout mouse models that closely resemble the null phenotype of the human disease have been generated by gene targeting (1-3). All of these hemophilia B mice have no detectable factor IX mRNA or protein and exhibit severe hemophilic phenotypes with spontaneous bleeding in the foot pads, spontaneous lethal internal bleeding, and nonstop bleeding after tail clipping. The mouse model systems accurately reflect the pathophysiology of human hemophilia B. Recombinant adenoviral vectors expressing murine or human factor IX upon injection in hemophilia B mice (129 and CD-1 strains) achieved only temporary correction of the bleeding diathesis because of the rapid formation of neutralizing antibodies to murine or human factor IX (2, 4). Although correction in the C57BL͞6 strain lasted Ͼ12 weeks without detection of antibodies to human factor IX (hFIX), the level of expression of factor IX declined during the time course of 12 weeks (4), possibly because of cytotoxic T lymphocyte response to adenoviral-transduced cells.Adeno-associated virus (AAV) is promising for in vivo gene therapy because the vector is based on a nonpathogenic virus and can infect both dividing and nondividing cells. Additionally, the vector is devoid of any viral coding sequences that reduce the risk of undesirable host immune responses. Several recent reports have demonstrated the persistent expression of transgenes in immunocompetent animals after delivery of AAV vectors into various tissues (5-11). And more recently, 1% of normal canine factor IX levels have been achieved in hemophilia B dogs by recombinant AAV (rAAV) vectors targeting liver or muscle (12,13). In this study, we explored the feasibility of...
Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.
Factors controlling proliferation of adrenocortical cells have been studied in Although several purified polypeptides have been reported to stimulate the proliferation of animal cells-in culture, a high degree of cell specificity has not been observed. Fibroblast growth factor (FGF) is mitogenic for fibroblast, adrenocortical, myoblast, smooth muscle, chondrocyte, vascular endothelial, granulosa, and luteal cells, whereas epidermal growth factor (EGF) is mitogenic for fibroblast, corneal epithelial, certain mammary epithelial, and granulosa cells (1). Several pituitary polypeptides long considered trophic for specific target cells in ivo either inhibit DNA synthesis when added directly to target cells in culture or have no effect on growth (2-6). Understanding of the mechanisms controlling organ-specific growth is, therefore, incomplete; interaction of several growth factors and hormones may be involved (7).A recently developed strain of bovine adrenocortical cells in culture has been used to study factors controlling proliferation of the adrenal cortical cell (8). FGF but not EGF stimulates proliferation of these cells, whereas corticotropin (ACTH) and prostaglandin El (PGE1), which stimulate cyclic AMP formation, inhibit proliferation (8, 9). Corticotropin and PGE1 both stimulate steroidogenesis and induce the steroid biosynthetic pathway. The present studies indicate that angiotensin II and III at low concentrations stimulate DNA synthesis as well as steroidogenesis in adrenocortical cells. The growth-stimulatory effect of angiotensin appears specific because several cell types, including cells reported to possess angiotensin II receptors, do not increase DNA synthesis with addition of angiotensin II (Schwarz/Mann) was routinely added to cultures 12 hr after addition of growth stimuli. Twelve hours later, medium was removed and 1 ml of a 1% aqueous solution of Triton X-100 was added. The cells were incubated with this solution for 5 min and the entire contents of the plate
The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) increases the growth rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (15 hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma factors is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM res nd to plasma factors, it is likely that the close contact of the celIs with the ECM restores their sensitivity to physiological factors present in plasma. Culture of most cells in vitro requires the presence of serum (1). Consequently, investigators have spent much effort to identify the various factors in serum that stimulate cell growth in vitro. An important step in the search for serum growth factors has been the finding that one of the most potent mitogenic factors present in serum is derived from platelets. Such a possibility, first postulated by Balk (2), was based on studies of the growth of chicken embryo fibroblasts in medium supplemented with plasma or serum. Chicken fibroblasts do not proliferate in plasma-containing medium (2), but when they are exposed to serum they proliferate actively. It was therefore concluded that serum contained growth-promoting activity that was absent from plasma (3). These studies were followed by reports which demonstrated that platelets are the source of a potent mitogen present in serum but not in plasma. Whereas plasma was unable to support the growth of aortic smooth muscle cells (4) or of BALB/c 3T3 cells (5), serum made from the same pool of blood stimulated their proliferation. Addition of a platelet extract to cell-free plasma-derived serum restored the growth-promoting activity (4-6). One could therefore conclude that one of the principal mitogens responsible for the induction of DNA synthesis present in whole blood serum is derived from platelets (4-6). The difference in the proliferative ability of cells exposed to plasma or to serum results from the absence of the platelet factor in the former.All studies have thus far used cells maintained on plastic rather than on a basal lamina or an extracellular matrix (ECM), which in vivo is the natural substate upon which cells migrate, proliferate, and differentiate. This difference in the substrate upon which the cells are maintained could have prevented their response to p...
The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation. In this study we demonstrate that substrate-bound laminin causes time-and concentration-dependent adherence of human platelets to the substrate. The binding of platelets to laminin was found to be similar in some respects, but different in others, to their binding to surfaces coated with fibronectin or collagen. The binding of platelets to laminin or fibronectin was not associated with their activation under conditions in which type I collagen activates the platelets as measured by [14C]serotonin secretion. Platelets bound to laminin and fibronectin differed in their appearance; they remained rounded on laminin whereas they flattened completely on fibronectin. Binding of platelets to fibronectin, but not laminin, is inhibited by a recently described peptide (Pierschbacher, M., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) containing the cell-attachment tetrapeptide sequence of fibronectin, which suggests that separate receptors exist for laminin and fibronectin. These studies establish laminin as a platelet-binding protein and suggest that laminin can contribute to the adhesiveness of exposed tissue matrices to platelets. Since laminin and fibronectin do not activate platelets, whereas collagen does, and laminin differs from fibronectin in that it does not induce spreading of the attached platelets, all three proteins appear to confer different signals to the platelets. Some of these may be related to platelet functions other than those necessary for the formation of a hemostatic plug.
The influence of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on the proliferation of cultured human fetal adrenal cells has been examined. Separated human definitive zone and fetal zone adrenal cells plated at low density in the presence of 10% serum and maintained on plastic culture dishes proliferated slowly. If the cultures were exposed to either FGF or EGF, the growth rate of the cells from each zone increased significantly. Half-maximal stimulation of cell proliferation for both zones occurred at a concentration of 3 X 10(-11) M for EGF and 8 X 10(-9) M for FGF. In addition, 125I-labeled EGF binding to both definitive and fetal zone cells demonstrated high affinity (Kd = 10(-9) M). To investigate the influence of an extracellular matrix (ECM) on cell proliferation, separated fetal adrenal cells maintained on plastic culture dishes were compared with cells maintained on a recently described ECM prepared from bovine corneal endothelial cells. Fetal adrenal cells maintained on the ECM had a significantly higher growth rate than cells maintained on plastic alone. These results demonstrate 1) the mitogenic role of EGF and FGF for human fetal adrenal cells, and 2) that the type of substrate upon which fetal adrenal cells are maintained has a profound influence on their proliferation.
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