Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac

Brain natriuretic peptide (BNP) gene expression and chronic activation of the sympathetic nervous system are characteristics of the development of heart failure. We studied the role of the beta-adrenergic signaling pathway in regulation of the human BNP (hBNP) promoter. An hBNP promoter (-1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal cardiac myocytes, and luciferase activity was measured as an index of promoter activity. Isoproterenol (ISO), forskolin, and cAMP stimulated the promoter, and the beta(2)-antagonist ICI 118,551 abrogated the effect of ISO. In contrast, the protein kinase A (PKA) inhibitor H-89 failed to block the action of cAMP and ISO. Pertussis toxin (PT), which inactivates Galpha(i), inhibited ISO- and cAMP-stimulated hBNP promoter activity. The Src tyrosine kinase inhibitor PP1 and a dominant-negative mutant of the small G protein Rac also abolished the effect of ISO and cAMP. Finally, we studied the involvement of M-CAT-like binding sites in basal and inducible regulation of the hBNP promoter. Mutation of these elements decreased basal and cAMP-induced activity. These data suggest that beta-adrenergic regulation of hBNP is PKA independent, involves a Galpha(i)-activated pathway, and targets regulatory elements in the proximal BNP promoter.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

AbstractWe delivered adenovirus vector (Ad) via intravitreous injection and monitored transgene (luciferase) expression in living mice (BALB/c) at multiple time points. In vivo live imaging technology was able to assess dynamically intraocular luciferase expression in a single animal population throughout the entire experiment period. Using this information, we were able to determine the optimal time point for readministration of Ad into the eyes and to dynamically study the time course of expression of a second Ad administration. Optical imaging demonstrated the limited period of transgene expression in eyes. Significant transgene signal was also detected in livers. The repeat intraocular delivery of the adenovirus resulted in significant blunting of transgene expression in both eyes and livers compared to the initial delivery. Periocular corticosteroid (triamcinolone acetonide) injection combined with initial Ad delivery was effective to rescue luciferase expression on repeat Ad vector delivery. However, this effect was not observed when corticosteroid was combined with repeat Ad delivery. Although corticosteroid enhanced ocular transgene expression, it also increased transgene expression in liver, which has potential safety implications. This dynamic transgene expression in eyes was successfully traced and monitored via a live imaging technique.     

Cardiac-specific gene expression facilitated by an enhanced myosin light chain promoter

BACKGROUND: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. METHODS: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. RESULTS: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc). DISCUSSION: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.     

Viral transgene expression delivered by repeat intraocular adenoviral vector injection: in vivo live imaging study

AbstractWe delivered adenovirus vector (Ad) via intravitreous injection and monitored transgene (luciferase) expression in living mice (BALB/c) at multiple time points. In vivo live imaging technology was able to assess dynamically intraocular luciferase expression in a single animal population throughout the entire experiment period. Using this information, we were able to determine the optimal time point for readministration of Ad into the eyes and to dynamically study the time course of expression of a second Ad administration. Optical imaging demonstrated the limited period of transgene expression in eyes. Significant transgene signal was also detected in livers. The repeat intraocular delivery of the adenovirus resulted in significant blunting of transgene expression in both eyes and livers compared to the initial delivery. Periocular corticosteroid (triamcinolone acetonide) injection combined with initial Ad delivery was effective to rescue luciferase expression on repeat Ad vector delivery. However, this effect was not observed when corticosteroid was combined with repeat Ad delivery. Although corticosteroid enhanced ocular transgene expression, it also increased transgene expression in liver, which has potential safety implications. This dynamic transgene expression in eyes was successfully traced and monitored via a live imaging technique.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma

BACKGROUND: Malignant gliomas remain refractory to Ad5-mediated gene therapy due to deficiency of the coxsackie adenovirus receptor on tumor cells. The purpose of this study was to evaluate whether changes in adenoviral tropism can enhance gene transfer in the context of malignant glioma. METHODS: We have identified several receptors that are over-expressed on tumor cells and created a series of pseudotyped Ad5 vectors that recognize these receptors: Ad5-RGD which binds alpha(v)beta3/alpha(v)beta5 integrins; Ad5/3 which contains adenovirus serotype 3 knob and binds to CD46; Ad5-Sigma which incorporates the reovirus sigma knob and binds to junctional adhesion molecule-1; and Ad5-pk7 which contains the polylysine motif and binds heparan sulfate proteoglycans. We also investigated the Ad5-CAV1 vector, which contains the knob of canine adenovirus type 1, a virus previously shown to infect glioma via an unknown mechanism. In this study, we compared these modified vectors for their ability to promote the expression of luciferase transgene both in vitro and in vivo. RESULTS: Our results indicate that all five modified vectors attained higher mean luciferase activity vs. control. Among them, Ad5-CAV1 and Ad5-pk7 attained the highest transduction efficiency independent of different tumor lines or infection time. Ad5-Sigma and Ad5-pk7 also demonstrated the least nonspecific infection in normal human astrocytes. Most importantly, Ad5-pk7 achieved 1000-fold increased transgene expression in human glioma xenografts in vivo. CONCLUSIONS: These results indicate that modifications of adenoviral tropism can enhance gene transfer in tumors that are poorly susceptible to adenoviral vectors and warrant further development of Ad5-pk7 for glioma gene therapy.     

Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes

We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or -myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca²⁺-ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations. cardiac myocytes; cell-specific expression; adenovirus vectors; calcium transport     

Integrin dependence of brain natriuretic peptide gene promoter activation by mechanical strain

Expression of the brain natriuretic peptide (BNP) gene in cultured neonatal rat ventricular myocytes is activated by mechanical strain in vitro. We explored the role of cell-matrix contacts in initiating the strain-dependent increment in human BNP (hBNP) promoter activity. Coating the culture surface with fibronectin effected a dose-dependent increase in basal hBNP luciferase activity and amplification of the response to strain. Preincubation of myocytes with an RGD peptide (GRGDSP) or with soluble fibronectin, each of which would be predicted to compete for cell-matrix interactions, resulted in a dose-dependent reduction in strain-dependent hBNP promoter activity. A functionally inert RGE peptide (GRGESP) was without effect. Using fluorescence-activated cell sorting, we demonstrated the presence of beta(1), beta(3), and alpha(v)beta(5) integrins in myocytes as well as non-myocytes and alpha1 only in non-myocytes in our cultures. Inclusion of antibodies directed against beta(1), beta(3), or alpha(v)beta(5), but not alpha(1), alpha(2), or cadherin, was effective in blocking the BNP promoter response to mechanical strain. These same antibodies (anti-beta(3), -beta(1), and -alpha(v)beta(5)) had a similar inhibitory effect on strain-stimulated ERK, p38 MAPK, and, to a lesser extent, JNK activities in these cells. Cotransfection with chimeric integrin receptors capable of acting as dominant-negative inhibitors of integrin function demonstrated suppression of strain-dependent BNP promoter activity when vectors encoding beta(1) or beta(3), but not beta(5), alpha(5), or a carboxyl-terminal deletion mutant of beta(3) (beta(3)B), were employed. These studies underscore the importance of cell-matrix interactions in controlling cardiac gene expression and suggest a potentially important role for these interactions in signaling responses to mechanical stimuli within the myocardium.     

Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes

Adenovirus-mediated transfer of cDNA encoding the chickenskeletal muscle sarco(endo)plasmic reticulumCa²⁺-ATPase (SERCA1) yieldedselective expression in cultured chick embryo cardiac myocytes undercontrol of a segment (268 base pair) of the cell-specificcardiac troponin T (cTnT) promoter or nonselective expression inmyocytes and fibroblasts under control of a constitutive viral[cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown toexpress transgenic SERCA1 ATPase. Expression was targeted tointracellular membranes and was recovered in subcellular fractions witha pattern identical to that of the endogenous SERCA2a ATPase. Relativeto control myocytes, transgenic SERCA1 expression increased up to fourtimes the rates of ATP-dependent (and thapsigargin-sensitive) Ca²⁺ transport activity of cellhomogenates. Although the CMV promoter was more active than the cTnTpromoter, an upper limit for transgenic expression of functional enzymewas reached under control of either promoter by adjustment of theadenovirus plaque-forming unit titer of infection media. CytosolicCa²⁺ concentration transients andtension development of whole myocytes were also influenced to a similarlimit by transgenic expression of SERCA1 under control of eitherpromoter. Our experiments demonstrate that a cell-specific proteinpromoter in recombinant adenovirus vectors yields highly efficient andselective transgene expression of a membrane-bound and functionalenzyme in cardiac myocytes.

     

Preservation of myocardial function after adenoviral gene transfer in isolated myocardium

Adenoviral gene transfer to the heart represents a promising model for structure-function analyses. Rabbit hearts were subjected to an ex vivo perfusion protocol that achieves gene transfer in >90% of cardiac myocytes. Contractile function of isolated myocardial preparations of these hearts was then observed for 2 days in a recently developed trabecula culture system. In sham-infected hearts, the initial developed force (F(init)) (15.6 +/- 3.7 mN/mm(2); n = 12) did not change significantly after 48 h (17.0 +/- 1.9 mN/mm(2); P = 0.46). In adenovirus-infected preparations, F(init) (14.3 +/- 1. 8 mN/mm(2); n = 21) did not significantly differ from the control (P = 0.75) and was unchanged after 48 h (15.3 +/- 2.5 mN/mm(2); P = 0. 93). After 2 days of continuous contractions, we observed homogenous and high-level expression of the reporter genes LacZ coding for beta-galactosidase and Luc coding for firefly luciferase. Luciferase activity increased more than 2,500-fold from background levels of 8. 7 x 10(3 )+/- 5.0 x 10(3) relative light units (RLU)/mg protein (from hearts transfected with promotorless adenovirus with luciferase transgene construct AdNULLLuc, n = 5) to 23.4 x 10(6)+/- 11.1 x 10(6)RLU/mg protein (from hearts tranfected with adenovirus with Rous sarcoma virus promotor and luciferase transgene construct AdRSVLuc, n = 5) in infected myocardial preparations (P < 0.005). Our results demonstrate a new ex vivo approach to achieve homogenous and high-level expression of recombinant adenoviral genes in contracting myocardium without adverse functional effects.     

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.     

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.