Complement inhibitory therapeutics and xenotransplantation.

Once complement-mediated HAR has been inhibited, the full spectrum of cellular and antibody-mediated inflammatory and immune responses characteristic of acute and chronic rejection will need to be counter-manded. But the fact remains that if xenotransplantation is to become a clinical reality, a clinically relevant means of inhibiting complement activation will be required. Soluble complement receptor type 1 provides such a therapeutic option and an option where the dosing regimen is under the control of the physician and can be adjusted in response to the needs of the patient.

Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival in Dahl salt-sensitive hypertensive rats transgenic for human cholesteryl ester transfer protein.

The acceleration of atherosclerosis by polygenic (essential) hypertension is well-characterized in humans; however, the lack of an animal model that simulates human disease hinders the elucidation of pathogenic mechanisms. We report here a transgenic atherosclerosis-polygenic hypertension model in Dahl salt-sensitive hypertensive rats that overexpress the human cholesteryl ester transfer protein (Tg[hCETP]DS). Male Tg[hCETP]DS rats fed regular rat chow showed age-dependent severe combined hyperlipidemia, atherosclerotic lesions, myocardial infarctions and decreased survival. These findings differ from various mouse atherosclerosis models, demonstrating the necessity of complex disease modeling in different species. The data demonstrate that cholesteryl ester transfer protein can be proatherogenic. The interaction of polygenic hypertension and hyperlipidemia in the pathogenesis of atherosclerosis in Tg[hCETP]DS rats substantiates epidemiological observations in humans.

Fc and C3b receptors on pulmonary endothelial cells: induction by injury.

Receptors for the activated third component of complement and for the Fc portion of immunoglobulin G are not expressed by apparently normal bovine pulmonary endothelial cells, but are expressed when the cells are exposed to white cell lysates or are infected with influenza or cytomegalovirus. The unmasking of these latent receptors may contribute to the pulmonary inflammatory response characteristic of, for example, anaphylaxis and to those lung diseases characterized by the deposition of immune complexes.

Endothelial cells of bovine pulmonary artery lack receptors for C3b and for the Fc portion of immunoglobulin G.

Bovine pulmonary endothelial cells do not possess receptors for the 3b component of complement (C3b) or for the Fc portion of immunoglobulin G. The lack of these receptors may help explain the nonthrombogenic function of endothelial cells. Our findings rule out the possibility that endothelial cells participate in pulmonary immune complex disease through the binding of C3b or Fc fragments.

High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors.

By virtue of its immediate contact with the circulating blood, the endothelium provides an attractive target for retroviral vector transduction for the purpose of gene therapy. To see whether efficient gene transfer and expression was feasible, rabbit aortic endothelial cells were infected with three Moloney murine leukemia virus-derived retroviral vectors. Two of these vectors carry genes encoding products that are not secreted: N2, containing only the selectable marker gene neoR, and SAX, containing both neoR gene and an SV40-promoted adenosine deaminase (ADA) gene. The third vector, G2N, contains a secretory rat growth hormone (rGH) gene and an SV40-promoted neoR gene. Infection with all three vectors resulted in expression of the respective genes. A high level of human ADA expression was observed in infected endothelial cell populations both before and after selection in G418. G2N-infected rabbit aortic endothelial cells that were grown on a synthetic vascular graft continued to secrete rGH into the culture medium. These studies suggest that endothelial cells may serve as vehicles for the introduction in vivo of functioning recombinant genes.

Effects of aspirin and dipyridamole on the degradation of adenosine diphosphate by cultured cells derived from bovine pulmonary artery.

To improve understanding of the mechanisms by which ADP is degraded during passage through the pulmonary vascular bed, we examined cultured endothelial and smooth muscle cells of bovine pulmonmary artery for their abilities to metabolize [8-14C]ADP. ADP is rapidly converted to AMP and then to adenosine, hypoxanthine, and inosine. Inosine is the major metabolite produced by endothelial cells. Radioactivity (5-10%) is accumulated intracellularly primarily as ATP. Medium containing 50 micro M ADP incubated with endothelial cells rapidly loses its ability to aggregate platelets and becomes antiaggregatory under conditions in which prostacyclin is absent. The antiaggregatory activity is probably the result of accumulated adenosine. 10 micro M dipyridamole inhibits cellular uptake of radioactivity by greater than 90%, and inosine in the medium is largely replaced by adenosine. This is accompanied by increased anti-aggregatory activity of conditioned medium, which can be matched by authentic adenosine at the same concentration. 1 mM aspirin had no effect on the metabolism of ADP by endothelial cells. Our results suggest: (a) Metabolism of ADP during passage through the lung is mainly the result of endothelial ADPase. (b) ADP released from aggregating platelets can be converted to the antiaggregatory substance, adenosine. Dipyridamole may exert some of its antithrombotic actions by preventing the intracellular uptake of adenosine, thereby increasing its concentration near the site of thrombus formation. (c) The ability of the vessel wall to degrade ADP should not be compromised by the use of aspirin as an antithrombotic drug. (d) Endothelium may retain some of its antithrombogenicity when prostacyclin generation is impaired.

Stimulation of rat endothelial cell transforming growth factor-beta production by bleomycin.

This study examines the hypothesis that mediators from lung endothelial cells could promote lung collagen synthesis in pulmonary fibrosis. Since bleomycin induces pulmonary fibrosis in humans and animals, the effects of this drug on endothelial cells were examined. Endothelial cell conditioned media were prepared in the presence of various doses of bleomycin, and tested for their ability to stimulate lung fibroblast collagen synthesis. The results show a dose-dependent stimulation of endothelial cell secretion of collagen synthesis stimulatory activity by bleomycin, which peaked at a dose greater than or equal to 100 ng/ml. Stimulation was selective for collagenous protein synthesis. Gel filtration analysis showed most of the activity to reside in fractions with an estimated molecular mass range of 10-27 kD. The activity was inhibited by anti-transforming growth factor-beta (TGF-beta)antibody, but not by nonimmune control IgG. The presence of TGF-beta was confirmed using the mink lung epithelial cell assay. Northern blotting revealed significant increases in TGF-beta mRNA in bleomycin-stimulated endothelial cells. Thus in vitro stimulation of endothelial cells by bleomycin upregulates TGF-beta production, presumably by increased transcription. In view of the chemotactic and matrix synthesis stimulatory properties of this cytokine, such an increase in TGF-beta production may play an important role in bleomycin-induced pulmonary fibrosis.

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

Cell-cell contact mechanisms.

Exciting new findings link characteristic properties of the inflammatory process previously not linked functionally. For example, it is now clear that oxygen radicals and leukocyte adhesion are intimately related in a carefully transduced and orchestrated series of events that culminates in release of granule contents, but not before the leukocyte has safely transversed the vessel wall. In addition to defining separate heterocellular contacts and homocellular aggregation we must now consider equilibrium events that allow associations among cell-cell partnerships involving different cell types.

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists.

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.

Endothelial cell permeability to water.

We have calculated diffusional permeability coefficients for tracer water and for [14C]antipyrine in endothelial cells. With these values and those from studies in whole lungs we set a range for diffusional water permeability coefficients of the intact endothelium.

The osmotic permeability of isolated calf pulmonary artery endothelial cells.

Osmotic permeability coefficients, PF, for water in isolated calf pulmonary artery endothelial cells determined over the temperature range 41 to 20 degrees C are 311.10(-5) cm.s-1 at 37 degrees C and 159.10(-5) cm.s-1 at 20 degrees C. The value at 37 degrees C is close to that reported earlier for the diffusional permeability coefficient, PD. The PF/PD ratio is 1.0 at 37 degrees C. The PF values are within the range of values extrapolated for filtration permeability in pulmonary endothelium. The temperature dependence expressed as the activation energy is 7.2 kcal.mol-1. The product of hydraulic conductivity, Lp (or PF) and of viscosity changes in water is not constant from 37 to 20 degrees C. These results can be interpreted to indicate a similar pathway for water whether under diffusional or osmotic gradients.

The diffusional transport of water and small solutes in isolated endothelial cells and erythrocytes.

The diffusional permeability coefficients, PD, for tritiated water (3HHO) 14C-antipyrine (AP) and 14C-iodoantipyrine (IAP) in isolated calf pulmonary artery endothelial cells and dog erythrocytes are measured with the linear diffusion technique at 11.5, 15, 20 and 37 degrees C. The PD values for both cell populations follow the sequence 3HHO > IAP > AP at each of the temperatures. PD for water is higher in the erythrocyte compared to the endothelial cells. The differences in PD for AP and IAP in the erythrocytes and endothelial cells are not dramatic and are similar to the differences seen in comparing permeation of the same solute through bilayers of different composition. A comparison of the values of PD calculated for the endothelial cells with those for isolated capillaries and the structured endothelium in whole lungs validates the use of the isolated cells as models for the endothelial cells in situ. Incubation of the endothelial cells with cis-vaccenic acid or cholesterol produces a reduction in PD for water and antipyrine. These data are analyzed in terms of Stokesian and non-Stokesian diffusion. The interpretation which best accommodates the data is that the phospholipid area of the membrane, rather than the hydrocarbon core, provides the greatest resistance to permeation for these solutes.

Tissue culture reduces Ia antigen-bearing cells in rat islets and prolongs islet allograft survival.

Prolongation of rat pancreatic islet allograft survival by a prior 7-day period of tissue culture was demonstrated, confirming previous reports by others. We then sought to identity those cells in islets capable of stimulating allograft rejection (Ia antigen-bearing cells) and to determine whether such cells and/or their Ia antigens might be reduced by tissue culture. Freshly isolated and 7-day-cultured Wistar-Furth rat islets were incubated with a mouse anti-rat Ia nonpolymorphic monoclonal antibody, then with peroxidase-conjugated goat anti-mouse antibody, and processed for electron microscopy. Peroxidase (Ia)-positive lymphocytes, macrophages, and capillary endothelial cells were identified in fresh but not in cultured islets. A radioligand assay, using 125I-protein A, revealed a 45% decrease in binding of Ia antibody to cultured compared with fresh islet cells. We conclude that Ia antigen-bearing lymphocytes, macrophages, and capillary endothelial cells in rat islets are reduced by tissue culture and that this may account, at least in part, for the decreased immunogenicity of cultured islet allografts.

Potentiation of endogenous nitric oxide with superoxide dismutase inhibits platelet-mediated thrombosis in injured and stenotic arteries.

OBJECTIVES: We tested the hypothesis that dismutation of superoxide anion increases endogenous levels of nitric oxide, resulting in inhibition of cyclic variations in blood flow in arteries that are injured and stenotic. BACKGROUND: Platelet adhesion and aggregation leading to cyclic flow variations might result, in part, from generation of superoxide anion that can deplete endogenously produced nitric oxide. METHODS: Spontaneous cyclic flow variations, monitored with a proximal Doppler probe, were induced in the carotid artery of anesthetized rabbits by clamping the vessel with forceps and placing a high grade stenosis at the site of injury. Bovine copper/zinc superoxide dismutase (12 mg/kg body weight, n = 5), a synthetic low molecular weight mimetic (12 mg/kg, n = 8) or buffer vehicle (n = 8) was administered intravenously as divided boluses over 45 min, and the frequency of cyclic flow variations was monitored for 4 h. RESULTS: Cyclic flow variations remained stable for 4 h in vehicle-treated animals (15 +/- 1 [mean +/- SEM]/30 min at baseline and 16 +/- 1/30 min after 4 h, n = 8) but exhibited a marked and persistent reduction in animals given copper/zinc superoxide dismutase (from 14 +/- 1/30 min at baseline to 4 +/- 1/30 min after 4 h) or the mimetic (from 15 +/- 1/30 min at baseline to 3 +/- 1/30 min after 4 h, p < 0.005). They were restored in three of four mimetic-treated animals during infusion of NG-monomethyl- L-arginine (100 mg/kg), an inhibitor of nitric oxide production. In addition, levels of cyclic guanosine 5'-monophosphate in platelets were elevated after administration of the mimetic (from 2.4 +/- 0.5 fmol/10(6) platelets at baseline to 4.9 +/- 0.6 fmol/10(6) platelets 45 min after the mimetic, p < 0.03, n = 6), whereas mean arterial blood pressure was decreased and flow velocity in the carotid artery was increased consistent with mediation of the effect on cyclic flow variations by increased endogenous nitric oxide. CONCLUSIONS: Dismutation of superoxide anion appears to attenuate platelet thrombus formation at a site of vessel injury by potentiation of endogenously produced nitric oxide. This approach may have utility to inhibit platelet-rich thrombosis in injured and stenotic arteries where production of superoxide anion is increased.

Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Studies on the mechanisms involved in antigen-evoked pleural inflammation in rats: contribution of IgE and complement.

Immunoglobulin E (IgE) has been shown to play a critical role in the allergic late-phase reaction, which is marked by intense leukocyte infiltration and edema. In this study we assessed the allergic pleural inflammation triggered by intrapleural (i.pl.) challenge in sensitized rats. We examined pleural effluent from actively sensitized rats following anti-IgE monoclonal antibody (mAb) (MARE-1) provocation for protein exudation, neutrophil as well as eosinophil accumulation. Inflammatory changes triggered by antigen after passive sensitization with IgE mAb was also assessed for comparison. Total serum level of IgE was found to be about threefold increased 7-8 days post-active sensitization, remaining augmented for at least 30 days. Increased levels of peritoneal leukocyte-bound IgE and serum IgE with specificity to ovalbumin were also detected. Nevertheless, the anti-IgE challenge in 14-day actively sensitized was shown to be a weak stimulus of neutrophil and eosinophil accumulation, despite being able to cause intense protein extravasation. Similarly, antigen challenge of IgE-passively sensitized rats caused protein leakage that was comparable to that induced by anti-IgE mAb in actively sensitized rats but led to a much lower neutrophil/eosinophil infiltration. Also, blockade of complement with recombinant human soluble C receptor-1 (sCR1) treatment prevented actively sensitized rats from reacting to antigen with neutrophil and eosinophil recruitment without modifying protein extravasation. These data suggest that IgE and complement-mediated mechanisms probably account for the exudation and leukocyte infiltration that is characteristic of the pleural inflammatory response observed in actively sensitized rats.

Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation.

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.

Vascular endothelial cell killing by combinations of membrane-active agents and hydrogen peroxide.

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.

Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival.

Hyperacute xenograft rejection may be modified by the activation and depletion of complement (C) using cobra venom factor (CVF). This method of prolonging xenograft survival is toxic and associated with systemic inflammation, which may potentially contribute to the pathologic features of delayed xenograft rejection. Soluble complement receptor type 1 (sCR1) inhibits both the classical and alternative C pathways and thus limits the production of proinflammatory products such as the anaphylatoxins. Hence, we investigated the effects of various sCR1 and CVF regimens, and combinations thereof, in the discordant guinea pig-to-Lewis rat cardiac xenograft model. Mean graft survival time (MST) was significantly prolonged with repetitive dosing (MST=22 hr) or continuous infusion of sCR1 (MST=32 hr) as compared with unmodified controls (MST=15 min). However, sCR1 did not prevent intragraft deposition of C3 or neutrophil infiltration and resulted in only partial inhibition of C-mediated hemolytic activity in vitro. Grafts in rats treated with a single dose of CVF (MST=67 hr) or repetitive doses of CVF (MST=69 hr) survived significantly longer than those treated with sCR1 alone, and lacked C3 deposition or neutrophil accumulation. Sera from these animals were completely depleted of C-mediated hemolytic activity. Animals treated with a single dose of CVF, or sCRI plus a single dose of CVF (MST=64 hr), had similar xenograft survival times. However, immunohistologic studies showed that addition of sCR1 to a single dose of CVF resulted in decreased macrophage activation and reduced levels of cytokines (tumor necrosis factor-alpha and interleukin-1beta) within xenografts as compared with that in recipients treated with CVF alone. Such decreased macrophage activation may result from the binding of C4b by sCR1, since combination therapy was associated with decreased intragraft C4b as compared with either therapy alone. High doses of sCR1 were well tolerated by rats and significantly prolonged discordant xenograft survival (MST=32 hr), although not to the same extent as CVF. The modification of the intragraft immune responses seen with CVF/sCR1 combination therapy may augment further therapeutic manipulations to achieve discordant xenograft survival without the attendant toxicity associated with repeated CVF administration.