Ana Z. Fernandez

Intravascular hemolysis increases atherogenicity of diet-induced hypercholesterolemia in rabbits in spite of heme oxygenase-1 gene and protein induction

Free radical mediated oxidation of apoB lipoproteins in the arterial intima appears to contribute to atherogenicity of the entrapped particles. A plausible pathogenic mechanism for oxidation is the one induced by heme leaking from erythrocytes that is then carried into the arterial wall by its high affinity for lipoproteins. In the intima, in the presence of H(2)O(2) secreted by macrophages, heme can be a potent oxidant. To study the role of heme as a promoter of oxidative stress damage in vivo we used a model of intravascular hemolysis (IVH) caused by phenylhydrazine in rabbits with and without diet-induced moderate hypercholesterolemia (MHC). Evaluation of the antioxidant status of plasma indicated that at the end of the treatment period this was compromised by the MHC-IVH. After 10 weeks the animals with combined MHC-IVH showed more of the aorta surface covered by lesions (27%+/-8, mean (SD) than the animals with only MHC (11%+/-7), in spite of having similar plasma levels of VLDL+LDL lipoproteins. The animals with only IVH, as well as the controls, showed minimal lesions (<1%). Heme oxygenase (HO-1) expression in aorta and other tissues was markedly increased in the group with MHC-IVH and it was correlated with the extent of IVH. The data suggest that the oxidative stress associated with IVH potentiates the atherogenicity of moderate hypercholesterolemia and that in spite of a strong induction of HO-1 this is not sufficient to counteract the atherogenicity of the combined condition.

Peroxisome Proliferator-Activated Receptors in the Modulation of the Immune/Inflammatory Response in Atherosclerosis

Inflammation has been recognized as an important hallmark of atherosclerosis. The pharmacological activation of PPAR-γ by the thiazolidinediones in diabetes, and of PPAR-α by the fibrates in hyperlipidemia has been shown to help to reduce inflammatory markers in preclinical and clinical studies. PPARs are known to modulate immune pathways through at least three different mechanisms: by direct binding to PPRE of anti-inflammatory cytokines genes; by transrepression of transcription factors like NF-κB and AP-1; or by corepression. The regulation of the inflammatory pathways by PPARs can be achieved on each one of the cells involved in the atherosclerotic process, that is, monocytes, macrophages, T cells, endothelial cells, and smooth muscle cells. Moreover, as each of these cellular components is interconnected with each other, PPAR activation in one cell type could affect the other ones. As activation of PPARs has clear ant-inflammatory benefits, PPARs ligands should be considered as a new therapeutical approach to ameliorate the exacerbated immune response in atherosclerotic diseases.

Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X

The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K(i)(K(d)) = 14.8+/-1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M(-1)s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K(d) obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces <14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors.

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation.

Abstract: The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p‐PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p‐PBMC with human platelets to assess the capacity of p‐PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 µg/ml), were performed with platelets in platelet‐rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p‐PBMC or human PBMC (h‐PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N‐(3‐ethyl‐1,5‐pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p‐PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p‐PBMC or h‐PBMC. Aggregation of human platelets in PRP, induced by p‐PBMC, was inhibited when DAPA (100 μm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p‐PBMC to aggregate human platelets. No deposition of C3c fragments on p‐PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p‐PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.

Pig peripheral blood mononuclear cells are directly associated with the thrombotic microangiopathy that complicates the induction of chimerism in pig-to-baboon xenotransplantation

The infusion of large numbers of porcine cells into primates in order to induce specific immunologic tolerance by mixed hematopoietic chimerism, results in thrombotic microangiopathy that can be fatal. For this reason, it is important to study in vitro the interaction of primate endothelial cells with pig cells. We show that pig peripheral blood mononuclear cells (p‐PBMC) activate human endothelial cells (hECs) through direct contact. Thus, when endothelial cells are cultured in the presence of p‐PBMC, overexpression of VCAM‐1 and E‐selectin adhesion molecules occurs within 3 h of culture and continues for at least 9 h. The co‐culture of p‐PBMC and hECs also results in an important adhesion of human platelets to both types of cell. Thus, viewed with the microscope, platelets aggregate above the endothelial cells and also around the pig cells. We present data that suggest that the presence of p‐PBMC may be more important with regard to the increase of platelet adhesion to the endothelial cells than the activation alone of the cells. Our results also show that p‐PBMC, and not the activated endothelia or the culture supernatant of activated hECs, are able to activate the coagulation cascade because they are able to generate thrombin when added to defibrinated human plasma. Overall, these findings suggest that p‐PBMC are of primary importance for the development of the thrombotic disorders that occur in primates transplanted with pig progenitor cells.

Atherogenicity of Hypercholesterolemia in the Precense of Hemolysis in Spite of Heme Oxygenase-1 Induction

Atherosclerosis appears to be caused mainly by a focal and complex tissue response to apoB-containing lipoproteins (LDL, IDL, VLDL) deposition in the arterial intima (Camejo et al., 1998b; Williams and Tabas, 1995). The retention of apoBlipoproteins by proteoglycans of the arterial intima seems to provide the opportunity for enzymatic and non-enzymatic alterations of their lipid and protein moieties. This appears to cause formation of substances, mainly bioactive lipids that acting locally on arterial cells could initiate an inflammatory atherogenic response (Camejo et al., 1998b; Hurt-Camejo et al., 2001). Among the most studied potentially atherogenic modifications of lipoproteins are those caused by free radical-mediated reactions, probably taking place in the intima (Steinberg and Witztum, 1999). However, the origin of reactive oxygen species (ROS) that affect lipoproteins and cells in arteries still remains uncertain. The major cause for this is that agents, like transition metals, used as a model of pro-oxidant conditions in vitro do not oxidize lipoproteins in the presence of other plasma components that are known to exist in circulation and in the intima (Steinberg and Witztum, 1999). Heme is an iron-containing catabolic product of hemoglobin that has high affinity for human low density lipoprotein (LDL) in plasma and that is capable of oxidizing this particle in diluted plasma (Camejo et ah, 1998a; Tribble et al., 1995) Heme interacts with the lipid surface monolayer and with specific positively charged segments of apoB-100 in the LDL particle (Camejo et al, 1998a). Heme transported by apoB-lipoproteins can accumulate in the arterial intima and become an in situ generator of ROS.