Vascular endothelial growth factor restores corporeal smooth muscle function in vitro.

PURPOSE: The therapeutic use of vasculogenic growth factors has been successfully demonstrated in models of organ ischemia. We determined whether vascular endothelial growth factor (VEGF) would reverse corporeal smooth muscle dysfunction in the hypercholesterolemic rabbit model of erectile dysfunction. MATERIALS AND METHODS: A total of 36 New Zealand White rabbits were fed a normal (12) or 1% cholesterol (24) diet and treated after 6 weeks with 0.9 mg. VEGF or vehicle. At 6 weeks 24 rabbits received a single intracavernous dose and 12 received a single intravenous bolus of either drug. Ten days after injection corporeal smooth muscle function was analyzed after relaxation to acetylcholine and sodium nitroprusside using isometric tension studies. Corporeal sections were assessed for smooth muscle content with f-actin staining and VEGF expression by immunohistochemical study and enzyme-linked immunosorbent assay. RESULTS: Endothelium dependent (acetylcholine) and nitric oxide mediated (sodium nitroprusside) smooth muscle relaxation were impaired in cholesterol fed animals (p = 0.021 and 0.003, respectively). Intracavernous VEGF treatment restored sodium nitroprusside mediated relaxation to normal (p = 0.015) and intravenous VEGF restored acetylcholine and sodium nitroprusside mediated relaxation (p = 0.014 and 0.018, respectively). Decreased smooth muscle content was noted in cholesterol fed animals versus normal diet controls (p = 0.008), which was not affected by VEGF treatment (p = 0.450). Corporeal endothelial cell content was increased after intracavernous but not intravenous VEGF treatment (p = 0.001 and 0.385, respectively). VEGF expression was augmented after treatment with recombinant VEGF (p <0.001). CONCLUSIONS: VEGF administration variably mitigated the impairment of corporeal smooth muscle relaxation in the hypercholesterolemic rabbit model of erectile dysfunction.

Adenovirus-mediated genetic manipulation of the myocardial beta-adrenergic signaling system in transplanted hearts.

OBJECTIVES: Ex vivo perfusion of the cardiac allograft during organ procurement is an ideal environment for adenoviral vectors with transgenes that target improving graft contractility. One such target is the beta-adrenergic receptor-signaling system, in which alterations in transgenic mice have elucidated novel means to improve the function of the heart in vivo. The purpose of the current study was to determine the functional consequences of beta-adrenergic receptor manipulation in a rabbit model of cardiac allograft transplantation. METHODS: New Zealand White rabbits weighing 3 kg served as recipients to 1-kg outbred donors. Donor hearts were arrested and harvested, and 1 of 3 adenoviral constructs was administered into the aortic root perfusing the graft. Transgenes delivered encoded either the human beta(2)-adrenergic receptor, a peptide inhibitor of beta-adrenergic receptor densensitization, or the marker transgene beta-galactosidase. RESULTS: Five days after cervical heterotopic transplantation, left ventricular performance was measured on a Langendorff apparatus. A moderate pattern of rejection was seen in all grafts. Biventricular myocyte expression of beta-galactosidase was observed, and beta(2)-adrenergic receptor density was elevated 10-fold in grafts that received adeno-beta(2)-adrenergic receptor. Left ventricular systolic and diastolic performance was significantly increased in grafts transfected with either adeno-beta(2)-adrenergic receptor or adeno-beta-adrenergic receptor densensitization compared with control grafts that received adeno-beta-galactosidase. CONCLUSIONS: Ex vivo adenovirus-mediated gene transfer is feasible in a rabbit allograft model and, more important, genetic manipulation of beta-adrenergic receptor signaling either by increasing beta(2)-adrenergic receptor density or blocking endogenous receptor desensitization improves graft function acutely in this allograft model.

Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance.

BACKGROUND: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. METHODS AND RESULTS: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. CONCLUSIONS: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Capillary density of skeletal muscle: a contributing mechanism for exercise intolerance in class II-III chronic heart failure independent of other peripheral alterations.

OBJECTIVES: The study was conducted to determine if the capillary density of skeletal muscle is a potential contributor to exercise intolerance in class II-III chronic heart failure (CHF). BACKGROUND: Previous studies suggest that abnormalities in skeletal muscle histology, contractile protein content and enzymology contribute to exercise intolerance in CHF. METHODS: The present study examined skeletal muscle biopsies from 22 male patients with CHF compared with 10 age-matched normal male control patients. Aerobic capacities, myosin heavy chain (MHC) isoforms, enzymes, and capillary density were measured. RESULTS: The patients with CHF demonstrated a reduced peak oxygen consumption when compared to controls (15.0+/-2.5 vs. 19.8+/-5.0 ml x kg(-1) x min(-1), p <0.05). Using cell-specific antibodies to directly assess vascular density, there was a reduction in capillary density in CHF measured as the number of endothelial cells/fiber (1.42+/-0.28 vs. 1.74+/-0.35, p = 0.02). In CHF, capillary density was inversely related to maximal oxygen consumption (r = 0.479, p = 0.02). The MHC IIx isoform was found to be higher in patients with CHF versus normal subjects (28.5+/-13.6 vs. 19.5+/-9.4, p <0.05). CONCLUSIONS: There was a significant reduction in microvascular density in patients with CHF compared with the control group, without major differences in other usual histologic and biochemical aerobic markers. The inverse relationship with peak oxygen consumption seen in the CHF group suggests that a reduction in microvascular density of skeletal muscle may precede other skeletal muscle alterations and play a critical role in the exercise intolerance characteristic of patients with CHF.

Transmyocardial laser revascularization limits in vivo adenoviral-mediated gene transfer in porcine myocardium.

OBJECTIVE: Transmyocardial laser revascularization (TMR) is emerging as a potential treatment option for patients with end-stage CAD, and adjuvant gene therapy may be helpful in further improving the results of the procedure. However, the effects of TMR on gene transfer are unknown. METHODS: Swine underwent left thoracotomy. TMR was performed to create five channels at 2-cm intervals in the anterolateral free wall of the left ventricle (LV) followed by injection of 1 x 10(9) plaque-forming units (pfu) of a replication-deficient adenovirus vector carrying the reporter gene beta-galactosidase (Ad.Pac beta-gal). An additional five direct injections of 1 x 10(9) pfu Ad.Pac beta-gal were made at 2-cm intervals in the posterolateral LV of each heart. Control animals underwent TMR alone/vehicle alone (n = 3) or empty virus alone/no treatment (n = 3) of the anterolateral/posterolateral LV, respectively. RESULTS: ELISA revealed significantly greater transgene expression in the direct Ad.Pac beta-gal injection versus TMR plus Ad.Pac beta-gal inject regions at both 3 (n = 6) (273.0 +/- 58.5 vs. 133.4 + 28.1 pg beta-gal/g protein, P = 0.02) and 7 days (n = 6) (180.0 + 59.9 vs. 56.7 + 18.1 pg beta-gal/g protein, P = 0.02) postoperatively. At 14 days postoperatively (n = 2), no transgene expression was detected in either region. No transgene expression was detected in any of the control regions at 3 days postoperatively. CD-18 staining revealed significantly greater inflammation in the TMR plus Ad.Pac beta-gal and TMR alone regions as compared to Ad.Pac beta-gal or vehicle (P < 0.001). CONCLUSIONS: Adenoviral-mediated gene transfer in conjunction with TMR is possible, although TMR appears to limit the degree of transgene expression attained as compared to direct intramyocardial injection alone, likely due to the greater immune response observed with the former. These findings may have important implications for therapeutic strategies aimed at combining TMR with gene therapy for CAD.

Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia.

BACKGROUND: The mechanism of clinical improvement after transmyocardial laser revascularization (TMR) is unknown. One hypothesis holds that TMR causes increased myocardial perfusion through neovascularization. This study sought to determine whether angiogenesis occurs after TMR in a porcine model of chronic myocardial ischemia. METHODS: Six miniature pigs underwent subtotal left circumflex coronary artery occlusion to reduce resting blood flow to 10% of baseline. After 2 weeks in the low-flow state, dobutamine stress echocardiography and positron emission tomography were performed to document ischemic, viable myocardium. The animals then underwent TMR and were sacrificed 6 months later for histologic and immunohistochemical analysis. RESULTS: Histologic analysis of the lased left circumflex region demonstrated many hypocellular areas filled with connective tissue representing remnant TMR channels. Histochemical staining demonstrated a highly disorganized pattern of neovascularization consistent with angiogenesis located predominantly at the periphery of the channels. Immunohistochemical analysis confirmed the presence of endothelial cells within neovessels. Vascular density analysis revealed a mean of 29.2+/-3.6 neovessels per high-power field in lased ischemic myocardium versus 4.0+/-0.3 (p<0.001) in nonlased ischemic myocardium. CONCLUSIONS: This study provides evidence that neovascularization is present long term in regions of ischemic, viable myocardium after TMR. Angiogenesis may represent the mechanism of clinical improvement after TMR.

Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA.

To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.

Effect of late modulation of nitric oxide production on murine lupus.

MRL/MpJ-Faslpr (MRL-lpr) and New Zealand Black/ White (NZB/W) mice develop spontaneous autoimmune disease characterized by autoantibody production and glomerulonephritis that progresses in parallel with increasing systemic nitric oxide (NO) production. A previously published study from our laboratory indicated that oral administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (NMMA) before the onset of clinical disease significantly decreased renal and joint pathology in MRL-lpr mice. To characterize the effect of late modulation of NO production in murine SLE, we administered oral NMMA and/or restricted dietary arginine after disease onset in two murine models of SLE. When receiving combined NMMA and arginine restriction, MRL-lpr mice had reduced joint pathology scores and NZB/W mice had lower renal pathology scores than control mice. These results indicate that modulating NO production after the onset of disease diminishes disease severity in two models of SLE, although not as effectively as treating before disease onset.

Differences in V kappa gene utilization and VH CDR3 sequence among anti-DNA from C3H-lpr mice and lupus mice with nephritis.

To investigate the molecular properties of anti-DNA from lpr mice that express high levels of anti-DNA without immune-mediated nephritis, the sequences of VH and V kappa genes encoding 11 monoclonal anti-DNA antibodies derived from C3H-lpr/lpr (C3H-lpr) mice were studied. All of the C3H-lpr monoclonal anti-DNA bound single-stranded DNA while five also bound double-stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H-lpr anti-DNA revealed the use of VH genes that encode anti-DNA from the MRL-lpr/lpr and (NZB X NZW) F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the V kappa genes from C3H-lpr mice lacked significant identity with previously reported V kappa genes for anti-DNA from lupus models. These results indicate that anti-DNA from C3H-lpr mice differ from anti-DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti-DNA in these mice.

Modulation of renal disease in autoimmune NZB/NZW mice by immunization with bacterial DNA.

Preautoimmune New Zealand Black/White (NZB/NZW) mice immunized with Escherichia coli (EC) double standard (ds) DNA produce antibodies that bind mammalian dsDNA and display specificities similar to spontaneous lupus anti-DNA. Since calf thymus (CT) dsDNA fails to induce these antibodies, these results suggest a special potency of foreign DNA in inducing serological manifestations of lupus in a susceptible host. To assess the effects of DNA immunization on clinical manifestations in NZB/NZW mice, we measured renal disease and survival of mice immunized with either (a) EC dsDNA as complexes with methylated bovine serum albumin (mBSA) in adjuvant; (b) CT dsDNA with mBSA in adjuvant; (c)mBSA alone in adjuvant; or (d) unimmunized. After immunization with EC dsDNA, NZB/NZW mice developed significant levels of anti-dsDNA antibodies. Nevertheless, these mice had less proteinuria, nitrate/nitrite excretion, and glomerular pathology than mice immunized with either mBSA alone, CT dsDNA/mBSA complexes, or unimmunized mice. Survival of the EC dsDNA immunized mice was significantly increased compared with the other mice. Furthermore, immunization of mice after the onset of anti-DNA production and proteinuria stabilized nephritis and prolonged survival. The improvement in renal disease occurred despite the expression of autoantibodies that bound mammalian dsDNA as well as glomerular antigens. These results suggest that bacterial DNA has immunological properties that attenuate murine lupus despite the induction of pathogenic antibodies.

The influence of variable-region somatic mutations on the specificity and pathogenicity of murine monoclonal anti-DNA antibodies.

Antibodies to DNA (anti-DNA) occur prominently in systemic lupus erythematosus and provoke inflammatory damage in the kidneys. To determine the factors that confer pathogenicity on antibodies of this specificity, we investigated the in vitro and in vivo glomerular binding by members of four clonally related sets of monoclonal anti-DNA antibodies from lupus mice. Somatic mutations within the clonal sets enhanced binding to double-stranded DNA (dsDNA). Binding to permeabilized glomeruli in vitro was observed among affinity-purified preparations of these antibodies independent of specificity for dsDNA. In normal mice injected with hybridoma cell lines, nephritis as assessed by histology and immunofluorescence did not correlate with antibody affinity for DNA. By multivariate analysis, in vitro glomerular binding was the most predictive parameter of histologic outcome. These findings indicate that somatic mutations occurring during maturation of the autoimmune response do not necessarily enhance pathogenicity.

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.

The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine.

MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.

Disease severity in rheumatoid arthritis: relationships of plasma tumor necrosis factor-alpha, soluble interleukin 2-receptor, soluble CD4/CD8 ratio, neopterin, and fibrin D-dimer to traditional severity and functional measures.

Rheumatoid arthritis is a complex inflammatory disease of unknown cause. Although various laboratory and clinical measurements are useful in managing these patients, there is a need for better tests to quantitatively assess disease activity. The purpose of this study was to investigate the association of certain immune and inflammation (I-I) parameters with four traditional disease severity measures and a functional measure in rheumatoid arthritis patients. A single set of patient blood samples was analyzed, and four traditional disease severity measures and patient functional statuses were determined from 64 consecutive outpatients with rheumatoid arthritis. Plasma tumor necrosis factor-alpha (TNF), soluble interleukin-2 receptor (sIL-2R), sCD4 and sCD8 (and the sCD4/sCD8 ratio), neopterin, and fibrin D-dimer were analyzed in relationship to Westergren erythrocyte sedimentation rate (ESR), physician assessment of disease activity, joint pain count, grip strength, and Arthritis Impact Measurement Scale (AIMS) scores. Rheumatoid arthritis patients had higher mean levels of all I-I measures (except sCD4) compared to healthy subjects. Initial significant correlations between TNF, sIL-2R, and D-dimer and several disease severity and functional measures were detected. When we controlled for the covariates age, gender, race, and medications, regression analyses indicated that, as a group, the I-I measures were significantly related to grip strength, physician disease severity rating, ESR, and total joint pain. When the predictive values of the I-I measures were tested controlling for the covariates and ESR, D-dimer was independently and significantly associated with variability in grip strength, physician disease severity, and AIMS physical disability, while TNF was associated with a significant amount of variability in total joint pain.(ABSTRACT TRUNCATED AT 250 WORDS)

Extravascular fibrin formation and dissolution in synovial tissue of patients with osteoarthritis and rheumatoid arthritis.

Fibrin deposition is a prominent finding in the synovium of patients with rheumatoid arthritis (RA). Macrophages are found in increased numbers in RA synovium, and these cells are known to produce a variety of procoagulant and anticoagulant molecules. Using immunohistologic techniques, the content and distribution of several important components of the coagulation system in the synovium of patients with RA, osteoarthritis (OA), or traumatic joint abnormalities requiring surgery were investigated. Samples from 3 patients from each category were examined in detail. RA synovium (compared with that of patients with OA or joint trauma) had increased numbers of macrophages and increased expression/content of fibrinogen, tissue factor, factor XIII, tissue transglutaminase, cross-linked fibrin (fibrin D dimer), urokinase-type plasminogen activator, and alpha 2-plasmin inhibitor. Macrophage content in RA synovium was increased in both the lining cell areas and the interstitial cell areas. Fibrinogen was distributed throughout the tissue in all samples and was greater in RA synovium. In trauma and OA synovia, tissue factor was seen only in association with vessels (endothelial cells), but in RA synovium, it was markedly increased throughout the tissues. While fibrin D dimer was seen in small amounts in synovial lining cell areas of trauma and OA synovia, it was present in increased amounts in the lining cell and interstitial cell areas of RA synovium. Factor XIII and tissue transglutaminase were present in scant amounts in trauma and OA synovia, but there were increased amounts of both (especially tissue transglutaminase) in RA synovium in the vessel, lining cell, and interstitial cell areas. Urokinase and alpha 2-plasmin inhibitor were also markedly increased in RA synovium. These results suggest that in inflamed synovium, there is ongoing extravascular tissue fibrin formation and dissolution that correlates with the degree of inflammation and macrophage content. Extravascular coagulation/fibrinolysis in RA represents a potential target for therapeutic intervention in this disease.

Vitronectin is a substrate for transglutaminases.

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.