Carlos A. Labarrere

Longitudinal cohort study on the effectiveness of lipid apheresis treatment to reduce high lipoprotein(a) levels and prevent major adverse coronary events.

Background We investigated in a longitudinal, multicenter, cohort study whether combined lipid apheresis and lipid-lowering medication can reduce extremely high levels of lipoprotein(a) (Lp[a]) and thus prevent major adverse coronary events (MACE) more efficaciously than lipid-lowering medication alone.Methods Eligible patients had coronary artery disease and Lp(a) levels ≥2.14 µmol/l (95th percentile). All patients received lipid-lowering medications alone until maximally tolerated doses were no longer effective, followed by combined lipid apheresis and lipid-lowering medication. The rates of the primary outcome, MACE, were recorded for both periods.Results A total of 120 patients were included. The mean duration of lipid-lowering therapy alone was 5.6±5.8 years, and that of apheresis was 5.0±3.6 years. Median Lp(a) concentration was reduced from 4.00 µmol/l to 1.07 µmol/l with apheresis treatment (P<0.0001); the corresponding mean annual MACE rate per patient was 1.056 versus 0.144 (P<0.0001).Conclusions Lowering of Lp(a) levels by apheresis was efficacious and safe, and we recommend this therapy for patients in whom maximally tolerated doses of medication alone have failed to control coronary artery disease-associated events.

Immunohistologic evidence that villitis in human normal term placentas is an immunologic lesion

Villitis of unestablished origin is a lesion in placentas from normal and high-risk pregnancies. We have studied villitis areas in 25 normal term placentas for immune cells, coagulation components, and endothelial markers. Villitis areas were filled with activated (HLA-DR, HLA-DP, and HLA-DQ reactive) macrophages. B lymphocytes were not identified, and T lymphocytes were of the helper (CD4) phenotype. Antibodies to coagulation components revealed perivascular and trophoblastic basement membrane deposits of factor IX, increased numbers of platelets, and fetal stem vessels that did not react with endothelial markers. These findings suggest helper T lymphocytes activate macrophages that mediate coagulation activation and alter endothelium. This combination of immunologic events results in tissue changes that are histologically diagnosed as villitis. It is not known what triggers these immunologic events, but the finding of villitis in normal placentas suggests the causative factor(s) is present in all pregnancies.

The complement system in human reproduction

ABSTRACT: Regulation of the complement system in reproduction is unique inasmuch as reproductive tissues represent the only condition where allogeneic interactions occur naturally. Both allogeneic extraembryonic membranes and semen that contact and interact with maternal cells and tissues must avert complement‐mediated damage to ensure reproductive success. Several regulators of complement activation exist. Membrane cofactor protein (MCP) and decay accelerating factor (DAF) inactivate C3 and C5 convertases on cell surfaces. In addition, CD59 inhibits the membrane attack complex (MAC) of the complement cascade. Strong expression of these membrane glycoproteins by trophoblast and amniotic epithelium has been observed. MCP, DAF, and CD59 likely safeguard extraembryonic tissues from complement damage originating from maternal and fetal blood or amniotic fluid.

Vascular endothelial growth factor expression in transplanted human hearts

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.

Antibodies to endothelial cells identify myocardial damage and predict development of coronary artery disease in patients with transplanted hearts

BACKGROUND Transplant-induced coronary artery disease is a leading cause of graft failure in cardiac allograft recipients after the first year of transplantation, but there presently is no test to identify patients at high risk for developing the disease. Our research is focused on development of a predictive test to identify patients at high risk of developing the disease. METHODS Sixty-eight cardiac allograft recipients transplanted and followed at Methodist Hospital between 1982 and 1996 were studied. Serial annual angiograms were used to diagnose coronary artery disease, and serial endomyocardial biopsies were used to detect cellular infiltrates and microvascular disease. Biopsy-matched serum samples were used for cardiac troponin-T determinations as measures of myocardial damage, and serum antibodies to endothelial cells were determined by using flow cytometry, enzyme-linked immunosorbent assay and immunoblotting techniques. The endothelial antibody data were evaluated statistically for associations with angiographic changes, biopsy findings and biochemical evidence of myocardial damage. FINDINGS Antibodies to endothelial cells were identified by all three techniques, and significant associations were found for the amount of antibody identified by Western immunoblotting with histological rejection grades in biopsies, which were confirmed immunocytochemically as macrophages (p<0.01) and T lymphocytes (P = 0.03). These antibodies also associated significantly with vascular antithrombin depletion (p = 0.02), biochemical evidence of myocardial damage (p = 0.005) and subsequent development of coronary artery disease (p = 0.03). INTERPRETATION The significant association of anti-endothelial antibodies with cellular infiltrates, depletion of vascular antithrombin and myocardial damage suggests a role for antibody in the development of transplant-induced arteriopathy. The significant association of antiendothelial antibodies with the future development of coronary artery disease further suggests that assessment of these antibodies may provide a non-invasive test to predict the development of transplant-induced coronary artery disease.

Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients. An immunocytochemical study.

BACKGROUND Tissue-type plasminogen activator (TPA) is the principal activator of plasminogen. Since hemostasis in the microcirculation of allografts is a well-recognized complication of transplantation, we asked (1) whether the distribution and amount of cellular TPA in biopsies of transplanted human hearts are associated with fibrin deposits in and around the microcirculation, (2) whether such changes involve the physiological inhibitors of TPA and plasmin, and (3) whether the presence of these activators and inhibitors of fibrinolysis in tissue is correlated with clinical outcome. METHODS AND RESULTS We immunocytochemically quantified the presence of fibrin, plasmin, TPA, and the TPA inhibitor PAI-1 in 938 biopsies from 68 consecutive cardiac allografts over a 54-month period. The localization, distribution, and quantification of TPA in arteriolar smooth muscle cells revealed that 35 of the 68 allografts maintained vascular TPA reactivity consistent with time-zero biopsies of autologous donor hearts: this was designated as the normal TPA group. In contrast, 33 of the 68 allografts significantly lost vascular TPA reactivity compared with time-zero biopsies of autologous donor hearts: this was designated as the depleted TPA group. Analysis of sequential biopsies from both groups during 54 months revealed that the mean cumulative quantitative TPA value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.9 +/- 0.02 (P = .0001), and the mean cumulative quantitative fibrin value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.5 +/- 0.05 (P = .0001). Biopsies of allografts in the depleted TPA group contained endothelial reactivity for TPA-PAI-1 complexes, whereas biopsies from the normal TPA group did not. Plasmin-associated molecules were rarely identified in biopsies of the normal TPA group but were present in the depleted TPA group, and the fibrin-to-plasmin ratio in the normal TPA group always was less than the fibrin-to-plasmin ratio in biopsies from the depleted TPA group. Analysis of demographic and risk factors revealed no significant differences between patients in the normal and depleted TPA groups, but none of the 35 patients in the normal TPA group died or were retransplanted, and 13 of the 33 patients in the depleted TPA group died or required retransplantation (P = .0001). CONCLUSIONS Time-zero hearts (n = 68) and 34 of 38 stable allografts contained immunocytochemically detectable TPA only in vascular smooth muscle cells. Twenty-nine of 30 patients with normal TPA in their time-zero biopsies who subsequently developed a poor clinical outcome were found to have depleted TPA in biopsies evaluated during their first postoperative month and remained depleted throughout the study. Of 33 patients with depleted TPA, 39% died or required retransplantation. Depleted arteriolar TPA associated significantly with vascular and interstitial deposits of fibrin, plasmin, and endothelial TPA-PAI-1 complexes. These findings indicate that hemostatic and fibrinolytic pathways are activated in falling allografts, and they reveal evidence of depleted TPA before clinical or histopathological signs of failure. Patients with such allografts were found to be at high risk of death independently of other widely used clinical/laboratory parameters of prediction.

Intercellular Adhesion Molecule‐1 (ICAM‐1) and HLA‐DR Antigens Are Expressed on Endovascular Cytotrophoblasts in Abnormal Pregnancies

PROBLEM: We asked if the lack of normal trophoblastic invasion of spiral arteries in the basal plate of abnormal pregnancies was associated with the expression of HLA‐DR antigens and intercellular adhesion molecules (ICAM‐1) on endovascular cytotrophoblasts.

Maternal Cells in Chorionic Villi From Placentae of Normal and Abnormal Human Pregnancies

PROBLEM: We asked if activated macrophages and CD4 positive T lymphocytes in placental chorionic villi with villitis were of maternal or fetal origin.

Pathologic markers of allograft arteriopathy: insight into the pathophysiology of cardiac allograft chronic rejection.

Transplant-associated coronary artery disease (CAD) is the principal limiting factor for the long-term survival of heart transplant patients. This review discusses early risk factors for the subsequent development of transplant-associated CAD. Early risk factors associated with a prothrombogenic microvasculature, such as deposition of microvascular fibrin, depletion of vascular tissue plasminogen activator, presence of endothelial activation of the allograft arterial tree, and loss of vascular antithrombin, as well as changes in circulation (ie, detectable serum cardiac troponin I and elevated serum soluble intercellular adhesion molecule-1 levels) are presented and discussed. New therapies that could improve the status of the allograft microvasculature and may prevent or mitigate the development of transplant-associated CAD are considered.

Suppressed hindlimb perfusion in Rac2−/− and Nox2−/− mice does not result from impaired collateral growth

While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.