Exogenous control of cardiac gene therapy: evidence of regulated myocardial transgene expression after adenovirus and adeno-associated virus transfer of expression cassettes containing corticosteroid response element promoters.

OBJECTIVE: Because of the relative inaccessibility of the heart for repeated gene therapy, it would be useful to regulate the expression of transgenes delivered in a single dose of a gene therapy vector. Incorporation into the vector of a regulatable promoter that is responsive to pharmacologic agents that are widely used and well tolerated in clinical practice represents such a control strategy. METHODS: A replication-deficient adenovirus or an adeno-associated virus containing a chimeric promoter composed of 5 glucocorticoid response elements and the murine thrombopoietin complementary DNA (AdGRE.mTPO or AAVGRE.mTPO) was administered to the hearts of Sprague-Dawley rats. Platelet levels were evaluated as a reporter of transgene activity with or without dexamethasone. For comparison, rats received a control adenovirus vector, AdCMV.mTPO or AdCMV.Null, and the control adeno-associated virus vector AAVCMV.luc, which encodes for the firefly luciferase (luc) gene. RESULTS: Platelet elevation in the AdGRE.mTPO group peaked 4 days after dexamethasone administration, with a return to baseline 1 week after the initial corticosteroid dose. Subsequent dexamethasone administration at 2 and 4 weeks resulted in similar but progressively decreased responses. The AAVGRE.mTPO group had 5 peak platelet levels to a minimum of 2.2-fold with respect to baseline without diminution with subsequent dexamethasone administrations out to 169 days. In contrast, the AdCMV.Null and AAVCMV.luc groups demonstrated no increase in platelet counts and the AdCMV.mTPO group demonstrated a slow rise to a single peak platelet count independent of dexamethasone administration. CONCLUSION: It may be possible to control on demand the expression of a gene transferred to the heart. This strategy should be useful in cardiac gene therapy.

The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling.

Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.

Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines.

Chemically modified tetracyclines [CMT-3 (IC50 approximately 6-13 microM = approximately 2.5-5 microg/ml) and CMT-8 (IC50 approximately 26 microM = 10 microg/ml), but not CMT-1, -2 or -5], which lack anti-microbial activity, inhibited nitrite production in LPS-stimulated macrophages. Unlike competitive inhibitors of L-arginine which inhibited the specific activity of inducible nitric oxide synthase (iNOS) in cell-free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA accumulation and protein expression in LPS-stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike hydrocortisone had no significant effect on murine macrophages transfected with iNOS promoter (tagged to a luciferase reporter gene) in the presence of LPS. However, doxycycline and CMT-3 augmented iNOS mRNA degradation, in LPS-stimulated murine macrophages. These studies show a novel mechanism of action of tetracyclines which harbours properties to increase iNOS mRNA degradation and decrease iNOS protein expression and nitric oxide production in macrophages. This property of tetracyclines may have beneficial effects in the treatment of various diseases where excess nitric oxide has been implicated in the pathophysiology of these diseases.

Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide.

Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage + cytokines + endotoxin. The superinduction of PGE2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor HN(G)-monomethyl-L-arginine monoacetate inhibited OA cartilage NO production by > 90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE2 production. Cytokine + endotoxin stimulation of OA explants increased PGE2 production above the spontaneous release. Addition of L-NMMA further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE2. These studies, together with others, also suggest that PGE2 may be differentially regulated in normal and OA-affected chondrocytes.

A novel mechanism of action of tetracyclines: effects on nitric oxide synthases.

Tetracyclines have recently been shown to have "chondroprotective" effects in inflammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1 beta-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > or = doxycycline inhibited both lipopolysaccharide- and interferon-gamma-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for beta-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of beta-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.