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R G Crystal

Publications and source records attributed to R G Crystal.

At least 37 records · Page 2Linked to original sources
PubMed2001

Protection against pulmonary infection with Pseudomonas aeruginosa following immunization with P. aeruginosa-pulsed dendritic cells.

To develop a Pseudomonas aeruginosa vaccine that allows the host immune system to select the antigens, we hypothesized that dendritic cells (DC) pulsed with P. aeruginosa would induce protective immunity against pulmonary infections with P. aeruginosa. Incubation of murine bone marrow-derived DC with P. aeruginosa in vitro led to uptake of P. aeruginosa and activation of the DC. Spleen-derived CD4(+) cells from mice immunized with P. aeruginosa-pulsed DC showed increased proliferation, demonstrating that DC pulsed with P. aeruginosa were capable of eliciting a P. aeruginosa-specific immune response. To evaluate if P. aeruginosa-pulsed DC can induce protective immunity against P. aeruginosa pulmonary infection, DC incubated with P. aeruginosa in vitro were administered systemically to syngeneic mice, and the mice were then challenged by intrapulmonary infection with P. aeruginosa (5 x 10(4) CFU/mouse) 13 days later. Unimmunized control mice and mice who had previously received naive DC or DC stimulated with lipopolysaccharide or Escherichia coli died within 72 h. In contrast, 45% of mice receiving P. aeruginosa-pulsed DC demonstrated prolonged survival (>14 days). Finally, DC-pulsed with heat-inactivated P. aeruginosa protected CD8(-/-) but not CD4(-/-) mice, demonstrating that CD4(+) T cells were required for the DC pulsed with P. aeruginosa to induce protective immunity.

Animals↗
PubMed2001

Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia.

In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.

3T3 Cells↗
PubMed2001

Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein.

The intracellular trafficking of adenovirus (Ad) subgroup B (e.g., Ad7) differs from that of subgroup C (e.g., Ad5) in that Ad5 rapidly escapes from endocytic compartments following infection whereas Ad7 accumulates in organelles. To assess the hypothesis that Ad7 is targeted to the lysosomal pathway, Ad7 and Ad5 were conjugated with fluorophores and their trafficking in A549 epithelial cells was analyzed by fluorescence microscopy. Within 1 h after infection, Ad7, but not Ad5, accumulated in the cytoplasm of A549 cells. The pH in the environment of Ad5 was nearly neutral (pH 7), while Ad7 occupied acidic compartments (pH 5) over the first 2 h with a gradual shift toward neutrality by 8 h. Ad7 partially colocalized with alpha(2)-macroglobulin and late endosomal and lysosomal marker proteins, including Rab7, mannose-6-phosphate receptor, and LAMP-1. The pH optimum for membrane lysis by Ad7, as well as a chimeric Ad5 capsid that expressed the Ad7 fiber (Ad5fiber7), was pH 5.5, while that for lysis by Ad5 was pH 6.0. Thus, the native trafficking pathway for Ad7 involves residence in late endosomes and lysosomes, with information encoded in the Ad7 fiber acting as a pH-dependent trigger for membrane lysis and escape to the cytosol.

Adenoviridae↗
PubMed2001

Rapid assessment of adenovirus serum neutralizing antibody titer based on quantitative, morphometric evaluation of capsid binding and intracellular trafficking: population analysis of adenovirus capsid association with cells is predictive of adenovirus infectivity.

Neutralizing antiviral antibodies are typically detected on the basis of inhibition of viral function, such as propagation of a viral infection or inhibition of viral gene expression. Evidence is presented that anti-adenovirus neutralizing antibodies can be evaluated by analysis of cell-associated capsids or by analysis of intracellular trafficking of the capsids within 1 h after infection. Quantitative analyses of these morphologic parameters represent rapid, broadly applicable, functional assays for the detection of anti-adenovirus neutralizing antibodies.

Adenoviridae↗
PubMed2001

Upregulation of transcription factors in lung in the early phase of postpneumonectomy lung growth.

In the adult rodent, pneumonectomy results in compensatory lung growth characterized by cell proliferation. The molecular mechanisms governing this response remain unknown. We hypothesized that, in the early period postpneumonectomy, upregulated expression of transcription factors drives the growth process. We utilized a cDNA expression array to screen for upregulated transcription factors after left pneumonectomy in adult C57BL/6 mice, using unoperated mice as controls. Quantification of mRNA expression in the remaining lung at 2 h demonstrated a twofold or greater upregulation of six transcription factors: early growth response gene-1 (Egr-1), Nurr77, tristetraprolin, the primary inhibitor of nuclear factor-kappa B (I kappa B-alpha), gut-enriched Krüppel-like factor (GKLF), and LRG-21. Northern analysis was used to quantify the upregulation of expression of these genes relative to sham thoracotomy and unoperated controls. The largest increase was in Egr-1 (4.7-fold > naive). Time-course analysis over the first 24 h confirmed the transient nature of the early upregulation. In the context that postpneumonectomy lung growth is associated with cell proliferation and that genes such as Egr-1, Nurr77, LRG-21, and tristetraprolin have known roles in stress response, vascular biology, embryology, and cellular development, these data support the concept that transcription factors function early in the cascade of events leading to the compensatory response.

Animals↗
PubMed2001

Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity.

Macrophage-derived chemokine (MDC) is a potent chemoattractant for antigen-specific T lymphocytes. We hypothesized that Adenovirus- (Ad-) transduced dendritic cells (DCs) overexpressing MDC would enhance the T cell-mediated humoral immune response specific for antigens presented by the DC. We challenged two strains of mice with lethal Pseudomonas aeruginosa infection 3 weeks after immunization with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa. MDC-expressing DCs specifically attracted T lymphocytes and preserved typical DC surface phenotypes without growth factors in vitro. Mice immunized with AdMDC/Pseudomonas/DCs developed high levels of serum anti-Pseudomonas Ab's and were protected from a lethal respiratory challenge with Pseudomonas. The in vivo protective immunity required CD4(+) T cells, B cells, and IL-4, but not CD8(+) T cells and IL-12. AdMDC/DCs pulsed with Pseudomonas yielded significant but not absolute cross-protection against different strains of P. aeruginosa. Pseudomonas-pulsed AdMDC/DCs protected mice from Pseudomonas but not Escherichia coli and vice versa; this microbe-specific protection correlated with microbe-specific induction of CD4(+) T cell proliferation and IL-4 secretion. Based on these observations, AdMDC-modified DCs pulsed with a killed bacteria may be a useful approach to vaccination against infectious disorders.

Adenoviridae↗
PubMed2001

Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.

BACKGROUND: Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells. With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium. MATERIAL AND METHODS: To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals. RESULTS: Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p < 0.0005). Western analysis of BAL fluid proteins showed the major VEGF isoform in respiratory epithelial lining fluid is VEGF165. CONCLUSIONS: With the knowledge that proteins of molecular mass like VEGF (34 to 46 kDa) slowly diffuse across the alveolar epithelium, it is likely that this high level "reservoir" of VEGF protein on the respiratory epithelial surface plays a role in normal lung endothelial biology. However, this compartmentalized VEGF reservoir may also be a "Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached.

Blotting, Western↗
PubMed2000

Tumor regression induced by intratumor administration of adenovirus vector expressing CD40 ligand and naive dendritic cells.

We have previously shown that in vivo genetic modification of tumors with an adenovirus (Ad) vector engineered to express CD40 ligand (AdmCD40L) induces tumor-specific CTLs and suppresses tumor growth in vivo. In the present study, we investigate the hypothesis that this treatment can be made more efficient with 10(2)-fold less Ad vector by also administering bone marrow-generated dendritic cells (DCs) to the tumor. Using AdmCD40L and the number of DCs that alone had no effect on tumor growth, the data show that the growth of CT26 (colon adenocarcinoma; H-2d) and B16 (melanoma; H-2b) murine s.c. tumors is significantly suppressed by direct administration of DCs into s.c. established tumors that had been pretreated with AdmCD40L 2 days previously. The antitumor effect produced by the combination therapy AdmCD40L + DCs correlated with in vivo priming of tumor-specific CTLs. The antitumor cell-mediated immunity was transferable to naive mice by spleen cells from AdmCD40L + DC-treated animals. The interactions between CD40L and CD40 within treated tumors were critical because tumor suppression was abrogated by coadministration to the tumors of neutralizing monoclonal antibody against CD40L along with the DCs. Finally, in vivo depletion and knockout mice experiments demonstrated that tumor regression produced by this combination therapy depends on CD8+ T cells, but not on CD4+ T cells. These findings should be useful in designing strategies for use of DCs and AdmCD40L in cancer immunotherapy.

Adenocarcinoma↗
PubMed2000

Evaluation of clinical parameters to predict Mycobacterium tuberculosis in inpatients.

BACKGROUND: Respiratory isolation has been recommended for all patients with suspected tuberculosis (TB) to avoid transmission to other patients and health care personnel. In implementing these guidelines, patients with and without TB are frequently isolated, significantly increasing hospital costs. The objective of this study was to derive a clinical rule to predict the need for respiratory isolation of patients with suspected TB. METHODS: To identify potential predictors of the need for isolation, 56 inpatients with sputum cultures positive for TB were retrospectively compared with 56 controls who were isolated on admission to the hospital based on clinically suspected TB but whose sputum cultures tested negative for TB. Variables analyzed included TB risk factors, clinical symptoms, and findings from physical examination and chest radiography. RESULTS: Multivariate analysis revealed that the following factors were significantly associated with a culture positive for TB: presence of TB risk factors or symptoms (odds ratio [OR], 7.9 [95% confidence interval (CI), 4.4-24.2]), a positive purified protein derivative tuberculin test result (OR, 13.2 [95% CI, 4.4-40.7]), high temperature (OR, 2.8 [95% CI, 1.1-8.3]), and upper-lobe disease on chest radiograph (OR, 14.6 [95% CI, 3.7-57.5]). Shortness of breath (OR, 0.2 [95% CI, 0.12-0.53]) and crackles noted during the physical examination (OR, 0.29 [95% CI, 0.15-0.57]) were negative predictors of TB. A scoring system was developed using these variables. A patient's total score of 1 or higher indicated the need for respiratory isolation, accurately predicting a culture positive for TB (98% sensitivity [95% CI, 95%-100%]; 46% specificity [95% CI, 33%-59%]). CONCLUSION: Among inpatients with suspected active pulmonary TB, a prediction rule based on clinical and chest radiographic findings accurately identified patients requiring respiratory isolation.

Adult↗
PubMed2000

Dendritic cells modified to express CD40 ligand elicit therapeutic immunity against preexisting murine tumors.

CD40 ligand (CD40L) is essential for the initiation of antigen-specific T-cell responses. This study is based on the hypothesis that dendritic cells (DCs) genetically modified ex vivo to express CD40L will enhance in vivo presentation of tumor antigen to the cellular immune system with consequent induction of antitumor immunity to suppress tumor growth. To examine this concept, subcutaneous murine tumors were injected with bone marrow-derived DCs that had been modified in vitro with an adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In B16 (H-2(b), melanoma) and CT26 (H-2(d), colon cancer) murine models, intratumoral injection of 2 x 10(6) AdmCD40L-modified DCs (CD40L-DCs) to established (day 8) subcutaneous tumors resulted in sustained tumor regression and survival advantage. This antitumor effect was sustained when the number of CD40L-DCs were reduced 10-fold to 2 x 10(5). Analysis of spleens from CD40L-DC-treated animals demonstrated that CD40L-DCs injected into the subcutaneous CT26 flank tumors migrated to the spleen, resulting in activation of immune-relevant processes. Consistent with this concept, intratumoral administration of CD40L-DCs elicited tumor-specific cytotoxic T-lymphocyte responses, and the transfer of spleen cells from CD40L-DC-treated mice efficiently protected naive mice against a subsequent tumor challenge. In a distant 2-tumor model of metastatic disease, an untreated B16 tumor in the right flank regressed in parallel with a left B16 tumor treated with direct injection of CD40L-DCs. These results support the concept that genetic modification of DCs with a recombinant CD40L adenovirus vector may be a useful strategy for directly activating DCs for cancer immunotherapy.

Adenocarcinoma↗
PubMed2000

Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture.

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P <.01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

ADP-ribosyl Cyclase↗
PubMed2000

Adenovirus vector-mediated gene transfer to regional lymph nodes.

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.

Adenoviridae↗
PubMed2000

Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis.

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.

Adenoviridae↗
PubMed2000

Free cholesterol enhances adenoviral vector gene transfer and expression in CAR-deficient cells.

Efficient adenovirus vector-mediated gene transfer depends on the presence of sufficient amounts of the high-affinity coxsackie-adenovirus (Ad) receptor (CAR) on the surface of the target cell leading to receptor-mediated endocytosis of the vector. The present study evaluates the effect of free cholesterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-deficient cells. Infection in the presence of free cholesterol at its maximum solubility in water led to increased binding, uptake, and expression of Ad in human skin fibroblasts and alveolar macrophages, two primary human cells known to be deficient in CAR. The effect of free cholesterol was maximal at its solubility maximum in aqueous solution. Increase of Ad vector-mediated gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CAR-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expression was dependent on coincubation of both cholesterol and Ad and was not dependent on the cholesterol content of the cell. Increased Ad vector-mediated gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad-cholesterol mixture showed complexation between Ad particles leading to formation of multivirus aggregates due to hydrophobic interaction. The addition of free cholesterol with Ad vectors may be a simple way to increase Ad-mediated gene transfer to cells that are poor targets due to their lack of a sufficient number of Ad receptors.

Adenoviridae↗
PubMed2000

Downregulation of CXCR4 gene expression in primary human endothelial cells following infection with E1(-)E4(+) adenovirus gene transfer vectors.

Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant.

Adenoviridae↗
PubMed2000

Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo.

To assess the biodistribution and pharmacokinetics of gene transfer vectors, real-time PCR with fluorescent TaqMan chemistry was used to quantify tissue levels of adenovirus gene transfer vectors (Ad) following myocardial administration. After optimizing the detection of the genome of Ad vectors expressing human vascular endothelial growth factor (Ad(GV)VEGF121.10) and Escherichia coli cytosine deaminase (Ad(GV)CD.10), a comparison was made of intramyocardial injection versus intracoronary delivery to the left ventricle of the pig. One hour post-intramyocardial administration, the left ventricular Ad genome level was 6.2 copies per cellular genome, 26-fold higher than the level of 0.24 copies per cellular genome following intracoronary administration. Relative to the vector levels after 1 h, the amount dropped 14- and 5.5-fold by 24 h following intramyocardial and intracoronary administration, respectively. Interestingly, the vector that escaped the left ventricle after intracoronary or intramyocardial administration to pigs was found primarily within the lung, an observation in marked variance to the biodistribution of Ad vector in rodents. In this regard, after intravenous injection to the pig, 90% of the recovered vector was found in the lung, and even after intrahepatic portal vein injection, 55% of the recovered vector was in the lung. These data have important implications regarding the use of experimental animals for safety studies on administration of Ad to humans.

Adenoviridae↗
PubMed2000

Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121.

BACKGROUND: Adenovirus (Ad) vector-mediated gene therapy strategies have emerged as promising modalities for the "biological revascularization" of tissues. We hypothesized that direct intramyocardial, as opposed to intracoronary, administration of an Ad vector coding for the vascular endothelial growth factor 121 cDNA (Ad(GV)VEGF121.10) would provide highly focal Ad genome levels, and increases in VEGF, ideal for inducing localized therapeutic angiogenesis. METHODS: Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial Ad(GV)VEGF121.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model. RESULTS: Intramyocardial delivery of Ad(GV)VEGF121.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial Ad(GV)VEGF121.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial Ad(GV)VEGF121.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration. CONCLUSIONS: These studies demonstrate that direct intramyocardial administration of Ad(GV)VEGF121.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human trials of gene therapy-mediated cardiac angiogenesis.

Adenoviridae↗