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C A Labarrere

Publications and source records attributed to C A Labarrere.

At least 37 records · Page 2Linked to original sources
PubMed1998

Expression of a novel high molecular-weight myosin light chain kinase in endothelium.

Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional gap formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca2+/calmodulin-dependent nonmuscle myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To characterize EC MLCK expression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase-polymerase chain reaction to demonstrate that freshly isolated and cultured human macro- and microvascular EC express only the EC MLCK isoform (214 kD), which is distinct from smooth-muscle MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated the presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in different cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzyme in endothelium, minimal level of expression in smooth muscle, and no expression in skeletal muscle. These data suggest that the novel 214-kD kinase, the only MLCK isoform found in endothelium, may be preferentially expressed in this nonmuscle tissue.

Animals↗
PubMed1997

Endothelial activation and development of coronary artery disease in transplanted human hearts.

CONTEXT: The development of coronary artery disease in heart transplants is often associated with graft failure. Early detection of allografts prone to develop this disease is essential to institute new therapeutic approaches that could prolong allograft function. OBJECTIVE: To determine if early activation of arterial/arteriolar endothelium predicts the development of coronary artery disease, graft failure, or both in transplanted human hearts. DESIGN: Prospective cohort study. SETTING: Heart Transplant Center. PARTICIPANTS: A total of 121 consecutive adult cardiac allograft recipients who received transplants between 1988 and 1995 and were followed up through 1996. MAIN OUTCOME MEASURES: Development of coronary artery disease and graft failure. METHODS: Immunocytochemistry was performed on serial endomyocardial biopsy specimens to evaluate endothelial activation markers (intercellular adhesion molecule-1 and histocompatibility antigen HLA-DR) in arteries and arterioles. The presence and progression of coronary artery disease was evaluated by annual coronary angiograms with side-by-side comparisons. RESULTS: None of the 121 donor hearts showed arterial/arteriolar endothelial activation before transplantation. Arterial/arteriolar endothelial activation was present in 78 and absent in 43 of 121 allografts during the first 3 months after transplantation. The time of appearance and the proportion of biopsy specimens showing endothelial activation during these first 3 months were significantly associated with the risk of developing coronary artery disease, the progression of the disease, and the time required to develop the disease (P<.001). Significantly more patients with arterial/arteriolar endothelial activation died or received a second transplant (P<.001). CONCLUSIONS: Activation of arterial/arteriolar endothelium in transplanted human hearts predicts development of coronary artery disease and increased risk of graft failure.

Adult↗
PubMed1995

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.

Adult↗
PubMed1995

Vascular tissue plasminogen activator and the development of coronary artery disease in heart-transplant recipients.

BACKGROUND: An aggressive and potentially fatal form of coronary artery disease may develop after cardiac transplantation. We studied the role of vascular tissue plasminogen activator (t-PA), the primary mediator of fibrinolysis, in the development of this problem. METHODS: We studied 78 consecutive recipients of cardiac allografts over a five-year period, and we collected follow-up data over a mean (+/- SE) of 32.5 +/- 2.0 months. The patients were studied with ventricular function tests, serial endomyocardial biopsies (16.6 +/- 0.5 per patient), and annual coronary angiography. Measurements of t-PA and its inhibitor were performed immunocytochemically on unfixed cryostat sections of endomyocardial-biopsy specimens with the use of monoclonal antibodies to t-PA and its inhibitor. RESULTS: In biopsy specimens obtained during the first three months of follow-up, 38 allografts had a normal distribution of t-PA in arteriolar smooth-muscle cells, whereas in 40 allografts there was depletion of t-PA that persisted in subsequent follow-up. Coronary artery disease developed during follow-up in 31 of 40 allografts (78 percent) with depletion of t-PA, but the disease developed in only 9 of the 38 allografts (24 percent) with normal t-PA levels (P < 0.001). Allografts with depletion of t-PA also had the t-PA inhibitor and were at greater risk for earlier and more severe disease than were allografts with normal arteriolar t-PA levels. Twelve patients whose allografts were depleted of t-PA either received a second transplant or died, whereas only one of the patients whose allografts had persistently normal t-PA levels died (P < 0.001). CONCLUSIONS: These findings reveal an association between the depletion of t-PA from arteriolar smooth-muscle cells and the subsequent development of coronary artery disease and decreased graft survival. Although we cannot be certain about a cause-and-effect relation, our data suggest a possible role for deficient fibrinolysis in the development of coronary artery disease in transplanted human hearts.

Coronary Angiography↗
PubMed1995

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. METHOD: The basal plates of placentae from 15 normal and 55 abnormal pregnancies, including preeclampsia, small-for-gestational age infants, and mothers with history of secondary recurrent spontaneous abortion, were studied immunocytochemically by using monoclonal antibodies to HLA-DR and ICAM-1. Spiral and uteroplacental arteries were identified by using a triple antibody technique with antibodies to cytokeratin, alpha-smooth muscle actin, and von Willebrand factor to detect cytotrophoblasts, arterial smooth muscle cells, and endothelium, respectively. RESULTS: Placentae with normal placentation showed the presence of uteroplacental arteries that contained endovascular cytotrophoblasts that were negative for HLA-DR and ICAM-1 antigens. Placentae from abnormal pregnancies showed the presence of spiral arteries without trophoblastic invasion and uteroplacental arteries that were surrounded by numerous macrophages and T lymphocytes. Endovascular cytotrophoblasts in uteroplacental arteries of placentae from abnormal pregnancies reacted with antibodies to HLA-DR and ICAM-1 antigens. CONCLUSION: Placentae from normal pregnancies show uteroplacental arteries that contain endovascular cytotrophoblasts that do not react with antibodies to ICAM-1 and HLA-DR antigens, and placentae from abnormal pregnancies with uteroplacental arteries that are associated with arteries that do not show physiological changes contain endovascular cytotrophoblasts that react with antibodies to ICAM-1 an HLA-DR antigens. Normal uteroplacental arteries were found to be not surrounded by round cell infiltrates, but uteroplacental arteries associated with arteries that lack physiological changes were surrounded by round cell infiltrates, indicating that round cell infiltrates and endovascular cytotrophoblasts which react with antibodies to ICAM-1 and HLA-DR antigens are associated with abnormal pregnancies. These findings suggest that the cellular infiltrates are associated with endovascular cytotrophoblasts that react with ICAM-1 and HLA-DR antigens.

Abortion, Habitual↗
PubMed1995

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. METHOD: We employed a double antibody immunocytochemical technique on placental sections from three normal and four abnormal pregnancies with small-for-gestational-age infants. All studied placentae were mismatched for the maternal-fetal HLA-DRw 52 antigen. Areas of immunopathology were identified by using a monoclonal antibody to a monomorphic determination on HLA-DR, and the origin of immunological cells in areas of immunopathology was identified by using a monoclonal antibody to a polymorphic determinant on HLA-DRw 52. RESULTS: We used a double antibody technique that employed monoclonal antibodies to HLA-DR and HLA-DRw 52 antigens and placentae that were mismatched for the maternal-fetal HLA-DRw 52 antigen. We found that the vast majority of immunological cells within villi with inflammation were of maternal origin. Quantitative studies showed that between 75 and 100% of the cells in normal as well as in abnormal pregnancies were of maternal origin, and that abnormal pregnancies had a significantly higher percentage of villi with maternal cellular infiltrates. CONCLUSION: Our data show unequivocally that cells in areas of placental immunopathology are predominantly of maternal origin, and that abnormal pregnancies are associated with significantly more villi containing immunological cells of maternal origin.

Adult↗
PubMed1994

Natural anticoagulant and fibrinolytic pathways in renal allograft failure.

This is an immunocytochemical study of the relationship between depletion of natural anticoagulant and fibrinolytic pathways and allograft survival following renal transplantation. Patients (n = 44) were classified in three groups according to the length of time between transplantation and allograft failure: group 1 (n = 14) failed within a month of transplantation; group 2 (n = 14) failed between one month and one year after transplantation; and group 3 (n = 16) failed after one year of transplantation. Control biopsies were from donor kidneys (n = 16) prior to transplantation. There were no statistically significant differences in recipient age, gender, donor kidney type (living-related versus cadaver), histocompatibility, and plasma cholesterol, triglycerides, or creatinine concentrations between groups. However, group 1 allografts had a greater depletion of the vascular heparan sulfate proteoglycan-antithrombin III natural anticoagulant pathway than allografts in group 2 or 3 (P < or = 0.05), and this depletion was associated with significantly greater fibrin deposition in group 1 than in either group 2 or 3 (P < or = 0.05). All three groups demonstrated severe depletion of tissue plasminogen activator from arteriolar smooth muscle cells and depressed fibrinolysis as evidenced by increased fibrin/plasmin ratios. However, no significant differences were found for either endothelial thrombomodulin or T cell, neutrophil, or macrophage infiltration between the groups. These data indicate that differences in graft outcome may be determined more by compromised vascular function than by the presence of cellular infiltrates.

Adult↗
PubMed1994

Antigenic identification of cells in spiral artery trophoblastic invasion: validation of histologic studies by triple-antibody immunocytochemistry.

OBJECTIVE: Trophoblastic invasion of spiral arteries in the placental bed is essential for normal human placentation. Because the absence of these physiologic changes is associated with abnormal pregnancies, we developed a simple immunocytochemical approach that allows simultaneous study of three cell types involved in spiral artery changes (i.e., trophoblastic, endothelial, and smooth muscle cells). STUDY DESIGN: Spiral and uteroplacental arteries in the basal plate of 70 term placentas were studied simultaneously with antibodies to keratin to identify cytotrophoblasts, alpha-smooth muscle actin to identify vascular smooth muscle cells, and von Willebrand factor to identify endothelial cells in 15 normal placentas and 55 placentas from abnormal pregnancies. RESULTS: Normal term placentas showed keratin-positive, alpha-smooth muscle actin-negative and von Willebrand factor-negative uteroplacental arteries. Most (37/55) placentas from abnormal pregnancies had keratin-negative, alpha-smooth muscle actin and von Willebrand factor-positive spiral arteries. Spiral artery atherosclerosis was keratin negative, alpha-smooth muscle actin positive, and von Willebrand factor positive and was identified in 22 abnormal and 0 normal placentas. CONCLUSION: Simultaneous identification of three principal cell types involved in human placentation allowed qualitative and quantitative studies of placentas from normal and abnormal pregnancies that validated previous histologic investigations and provided a new approach to detect spiral arterial changes.

Abortion, Habitual↗
PubMed1994

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.

Biopsy↗
PubMed1993

Predominant expression of the beta subunit of prolyl 4-hydroxylase (disulfide isomerase) in human extravillous trophoblasts.

Prolyl 4-hydroxylase is a heterodimeric enzyme that is crucial in the biosynthesis of collagen. The beta subunit of this enzyme is a multifunctional protein which is also known as protein-disulfide isomerase. Immunofluorescence and monoclonal antibody (Mab) 5B5 were used to localize the beta subunit in human extraembryonic tissues. The strongest sites of 5B5 reactivity were extravillous cytotrophoblasts in the basal plate, uteroplacental arteries and amniochorion, syncytiotrophoblast displayed variable weaker reactivity. Only a small fraction of placental 5B5 antigen was detected as a component of prolyl-4-hydroxylase by affinity chromatography on immobilized polyproline. The results indicate a difference in the expression of an endoplasmic reticulum marker between villous and extravillous trophoblast. The predominance of 5B5 antigen in extravillous trophoblast could be associated with an increased ability to synthesize collagen or other enzymatic reactions associated with prolyl 4-hydroxylase beta subunit.

Antibody Specificity↗
PubMed1993

Immunological studies of lactoferrin in human placentae.

Lactoferrin (LF) and transferrin (Trf) are glycoproteins with strong affinities for ferric ions. Human syncytiotrophoblastic membranes analyzed by enzyme linked immunosorbent assay (ELISA) and immunoblotting were negative with monoclonal and polyclonal antibodies to LF. Immunohistological studies of 35 normal placentae showed that LF was absent from the trophoblast basement membranes, stroma and fetal stem vessel endothelium, but positive cells were occasionally noted in intervillous spaces and fetal stem vessels. In contrast, many LF-positive cells were identified within areas of immunopathology identified by the presence of T cells, HLA-DR-positive macrophages and platelets. Double-antibody experiments showed that the LF-positive cells in these areas reacted with CD15 and CD16 monoclonal antibodies (mAbs), indicating that the cells were polymorphonuclear neutrophils (PMN). PMN from peripheral blood analyzed by flow cytometry and immunocytology also showed reactivities with anti-LF, CD15 and CD16 and we consistently found that circulating PMN reacted better than placental PMN with antibodies to MHC class I antigens and gp 100, (CD67), which is a neutrophil activation marker. PMN adherent within placentae had no detectable MHC class I or CD67 antigens. These findings suggest PMN adherent to placental tissues down-regulate or alter plasma membrane markers. LF appears to play a role in placental inflammation, for LF-positive cells were significantly enriched in areas of immunopathology.

Antibodies, Monoclonal↗
PubMed1993

Tissue plasminogen activator in human cardiac allografts.

The activation of hemostasis and fibrinolysis frequently is observed in allografted organs. Plasminogen is activated by urokinase and tissue plasminogen activator (tPA). We have studied human hearts before and after transplantation to determine if fibrin deposition within the microcirculation is associated with a depletion of myocardial tPA, and if such depletion of tPA is associated with decreased fibrinolysis. We found that tPA in pretransplanted hearts and in biopsies from hearts of most patients with a stable clinical course is confined to arterial and arteriolar smooth muscle cells. The depletion of smooth muscle cell reactivity was associated with microvascular fibrin deposition in unstable allografts, and the appearance of endothelial cell tPA reactivity heralded a bad prognosis. Successful medical management was signaled by a loss of endothelial tPA reactivity and a return of tPA reactivity in arterial and arteriolar smooth muscle cells. These findings indicate a central role for tPA in maintaining the integrity of the microcirculation in transplanted human hearts.

Biopsy↗
PubMed1993

Modulation of vascular antithrombin III in human cardiac allografts.

The natural anticoagulant pathway involving heparan sulfate proteoglycan and antithrombin III (ATIII) was studied in serial biopsies from 90 cardiac allograft recipients. The ATIII component of this pathway was identified immunocytochemically on venous endothelium and arterial smooth muscle cells and intima of normal donor hearts and stable allografts. Unstable grafts lacked vascular ATIII and contained fibrin deposits. Neither stable nor unstable grafts had ATIII-reactive capillary endothelium. Grafts with absent vascular ATIII could (1) result in death, (2) revert to an arterial/venous ATIII distribution or (3) develop ATIII-reactive capillary endothelium. The development of ATIII-reactive capillaries was associated with a survival advantage, and such reactivity seemed to be promoted by heparin.

Antithrombin III↗
PubMed1993

Expression of CD59, a human complement system regulatory protein, in extraembryonic membranes.

CD59 (leukocyte cluster of differentiation antigen 59), is a phosphatidylinositol glycan-anchored membrane protein that inhibits lysis of cells by terminal complement system components. To further define complement regulatory proteins relevant to pregnancy, this study characterized the expression of CD59 in human extraembryonic membranes. Immunohistology with CD59 monoclonal antibody MEM-43 showed that this molecule was normally present on the apical surface of the syncytiotrophoblast, on extravillous cytotrophoblast, and amniotic epithelium. Immunoblotting confirmed that first and second trimester syncytiotrophoblast microvilli (STM) contained a glycoprotein similar in mass and glycosylation to CD59 from adult cells and tissues. Reactivities of STM with MEM-43 in ELISA were 2- to 6-fold higher than those of kidney, erythrocyte and platelet membranes. Term placental STM from recurrent spontaneous aborting patients after immunotherapy, reacted with MEM-43 in ELISA similarly to STM from normal individuals. Plasmas from pregnant women and umbilical cords had 50% or greater reactivity with MEM-43 than did normal plasmas. CD59 could help protect extraembryonic epithelia from damage by complement in maternal blood and amniotic fluid. The apical location of CD59 reflects the immunological roles and functional polarization of plasma membranes in the syncytiotrophoblast.

Abortion, Spontaneous↗