Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells

We propose that a long-term cure for the recalcitrant chronic venous ulcer must involve a dual surgical approach including (1) wide excision of the ulcer and surrounding liposclerotic tissue bed, and (2) replacement by a free flap containing multiple, competent microvenous valves with a normal microcirculation. Advantages of free flaps over skin grafting include improvement of the underlying pathophysiology; increase in blood supply to the area; ability to cover exposed bone, joint, or tendon; and a lower incidence of recurrence. During the past 8 years, 20 consecutive muscle free flaps were performed in 18 patients for 19 recalcitrant venous ulcers (two "sequential" flaps to the ipsilateral leg in 1 patient and a repeat flap after initial failure in 1 patient). Twelve males and 6 females ranged in age from 17 to 76 years (mean, 44 years). Nontraumatic, nonosteomyelitic venous ulcers had been present for an average of 3.5 years (range, 1-10 years) and failed an average of 2.4 skin grafts (range, 0-6 grafts). Defects ranged from 100 to 600 cm2 (mean, 238 cm2). Donor tissues included rectus abdominis (N = 13), latissimus dorsi (N = 5), gracilis (N = 1), and serratus (N = 1) muscles. Recipient vessels included posterior tibial (N = 12), anterior tibial (N = 6), and peroneal (N = 2). In all instances except one, only one vein, usually one of the venae comitantes, was anastomosed in end-to-end fashion. Successful free tissue transfer was accomplished in 18 of 20 flaps (90%). Complications included infection with partial flap and/or skin graft loss (three flaps), and partial skin graft loss (two flaps). There were no recurrences within the flaps; however, breakdown occurred at the junction between the flap and residual adjacent liposclerotic skin in 1 patient. Follow-up average 32.7 months (range, 8-65 months); 3 patients were lost to follow-up. Free muscle transfer can provide a long-term cure for the recalcitrant venous ulcer by replacing the diseased tissue bed with healthy tissue containing multiple, competent microvenous valves and a normal microcirculation. This can be accomplished in one reconstructive procedure with excellent long-term results.     

Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor.     

Thrombin regulates PDGF expression in bovine glomerular endothelial cells

BACKGROUND: Hispanic populations have been shown to be at high risk for smoking. The complex psychological process of adaptation to a different culture (acculturation) has been linked to smoking among Hispanic adults and adolescents. Although a positive association between acculturation and smoking appears to depend on gender among adults, research with Hispanic adolescents has ignored the moderating effect of gender. METHODS: Students in 22 New York City schools completed self-report questionnaires and provided carbon monoxide breath samples at two annual assessments. Sixth and seventh graders who identified themselves as Hispanics participated in the study (N = 1,295 at baseline; N = 1,034 at 1-year follow-up). The questionnaire included items related to smoking, acculturation, and demographic characteristics. RESULTS: Analyses were conducted to determine the effects of linguistic acculturation and gender on smoking. Girls smoked more frequently than boys at both time points. Being more acculturated was also associated with more smoking at the two survey assessments. As predicted, adolescent smoking depended on both gender and linguistic acculturation. For girls, but not boys, the highly acculturated adolescents smoked more frequently than either the bilingual or the less acculturated. CONCLUSIONS: Based on these findings, smoking prevention programs designed for Hispanic youth may benefit from an emphasis on Hispanic culture.     

Thrombin potentiates volume-activated chloride currents in pulmonary artery endothelial cells

In bovine pulmonary artery endothelial cells, ionic currents and the concentration of free intracellular Ca2+ ([Ca2+]i) were measured with a combined patch clamp and Ca(2+)-fluorimetric method (Fura-2). Volume-activated Cl-currents (ICl,vol) were activated by a 13 or 28% decrease in tonicity. Thrombin, 1 U/ml, strongly potentiated ICl,vol preactivated by low hypotonicity (13% HTS) but had no effect on ICl,vol preactivated by stronger hypotonic challenges (28% HTS). The thrombin-induced potentiation was not affected by buffering [Ca2+]i at 50-100 nmol/l and omitting extracellular Ca2+. A peptide agonist of the thrombin receptor, SFLLRN, also potentiated ICl,vol, while an enzymatically inactive thrombin analogue, DIP-thrombin, was without effect. These result suggest that proteolytic activation of the thrombin receptor sensitises the activation of ICl,vol in endothelial cells in a Ca(2+)-independent mechanism.     

Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis

The thrombin thrombomodulin dependent activation of the plasma protein TAFI (Thrombin Activatable Fibrinolysis Inhibitor) and Subsequent Inhibition of Fibrinolysis by the TAFIa is described. Work to date indicates that TAFIa is a carboxypeptidase B enzyme that suppress fibrinolysis most likely by down regulating the cofactor functions of partially degraded fibrin. The existence of TAFI provides the explanation for the apparent profibrinolytic effect of activated protein C. and implies the existence of an explicit molecular connection between the blood coagulation of fibrinolytic cascades that is expressed through the thrombin thrombomodulin dependent activation of TAFI. Thus, thrombin generation can, in principle, result in the suppression of fibrinolysis.     

Soluble urokinase receptor from fibrosarcoma HT-1080 cells

A soluble form of urokinase-binding protein has been isolated from the human fibrosarcoma cell line HT-1080 and cell lines derived from it. Conditioned media of these cells were collected after overnight incubation under serum-free conditions, and were concentrated and passed through a column of Sepharose 4B to which high-molecular-weight urokinase had been attached. After thorough washing, a polypeptide could be eluted from the column with 1 M acetic acid. This material appeared to be a single band of approximately 60 kDa on SDS polyacrylamide gel. It cross-reacted with commercial antibodies made against urokinase receptor, and could be chemically cross-linked to the amino terminal fragment of urokinase. This material was similar to the urokinase receptor that was cleaved from HT-1080 cells by means of phosphatidylinositol-specific phospholipase C.     

Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor

The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.     

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells

The development and validation of family-based alternatives to out-of-home placements for children is an important goal in the mental health services field. The rigorous evaluation of such alternatives, however, can be difficult to accomplish. The purpose of this article is to describe initial barriers experienced during the pilot study of a randomized trial, funded by the National Institute of Mental Health, conducted in a field setting, and the strategies that were used to overcome these barriers. The randomized trial is examining home-based multisystemic therapy as an alternative to the psychiatric hospitalization of youths presenting psychiatric emergencies. The pilot study illuminated the interface of treatment and services research issues, prompting significant changes in the project's clinical procedures, organization, and supervisory processes, as well as in the project's interface with existing community resources for serving youths with serious emotional disturbances.     

Mercury-stimulated histamine uptake and binding in cultured astroglial and cerebral endothelial cells

BACKGROUND: The reference method for detecting specific Epstein-Barr virus (EBV) antibodies is indirect immunofluorescence (IF) with EBV-infected cells. The availability of protein purified from infected cells and more recently of recombinant polypeptides designed to contain immunodominant epitopes, has enabled the development of commercial enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of EBV infection. OBJECTIVE: Evaluation of ELISA-based EBV serodiagnosis in comparison with indirect immunofluorescence. STUDY DESIGN: We have first compared three commercial ELISA test systems with our in house indirect immunofluorescence assay for classifying correctly a set of serum samples into clinical categories (acute infection, past infection, interfering non-EBV infection, persistent infection). Additionally a prospective analysis with the best performing ELISA test (Enzygnost) was then carried out by running the ELISA test in parallel with the indirect immunofluorescence assay on 324 consecutive clinical samples sent to our laboratory for EBV serodiagnosis. RESULTS: For the serodiagnosis of past EBV infection and acute EBV infection all three commercial ELISAs performed well in comparison with indirect immunofluorescence. When testing samples positive for cytomegalovirus (CMV), Toxoplasma or herpes simplex IgM, interference in the IgM tests was observed with the three ELISAs. In some instances we could demonstrate that the positive IgM results were due to EBV reactivation. The observed discrepancies between ELISA and IF for the serodiagnosis of chronic EBV infection or EBV reactivation, point to the difficulty for the serodiagnosis of persistent EBV infection on single serum samples. According to our prospective study the EBV IgG determination was accurate. A positive IgM result was not always indicative of an acute infection. Positive IgM results due to EBV reactivation were observed. A positive EBV nuclear antigen (EBNA) IgG result in those samples precluded acute infection. CONCLUSIONS: 90-95% of samples could be classified correctly into clinical categories by a two parameter ELISA system detecting IgG and IgM against a standardized mixture of EBV antigens, allowing standardization and automation of EBV-specific serology. The absence of EBNA IgG was useful as a second line confirmatory assay for acute EBV infection.     

Autocrine/paracrine role of adrenomedullin in cultured endothelial and mesangial cells

Neuropsychological profiles were assessed in a large group of nondemented control subjects (n = 261) and individuals with dementia of the Alzheimer type (DAT) (n = 407) by subjecting their psychometric test results to a factor analysis. Nondemented control subjects were functionally homogeneous with only one factor accounting for the results. The results of the factor analysis on the very mild DAT and mild DAT groups, however, yielded a mental control/frontal factor, a memory-verbal/temporal factor, and a visuospatial/parietal factor. Forty-one of the original set of participants came to autopsy an average of 5.1 years after psychometric testing and had neurofibrillary tangles, total senile plaques, and cored senile plaques estimated from frontal, temporal, and parietal regions. The results of correlations indicated that the relative burden of cored senile plaques was systematically related to the three psychometric factors. These results suggest a connection between the specific functions as defined by neuropsychological measures and specific neuropathology occurring in associated areas of cortex.     

Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.     

Activation of cultured vascular endothelial cells by antiphospholipid antibodies

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.     

Hypoxia reoxygenation-induced injury of cultured pulmonary microvessel endothelial cells

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.     

Glycosyltransferase activities in cultured endothelial cells of bovine brain microvascular origin

We report two cases of salmonella osteomyelitis isolated to the pelvis in white adolescents aged 12 and 16 years. No underlying medical condition predisposed these children to salmonella osteomyelitis, and the clinical course was prolonged before definitive diagnosis. The key to diagnosis and the localization of the site of the pathologic condition was made from radionuclide studies performed 2-3 weeks from the onset of symptoms. Clinicians should be aware of isolated salmonella osteomyelitis of the pelvis in normal children, especially when imaging studies are normal at initial presentation. Technetium-labeled bone scans may be normal < or = 2 weeks from the onset of symptoms. Definitive diagnostic testing should include a gallium scan and computed tomography scan when technetium bone scans are negative. Treatment with antibiotics alone is successful.     

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells

OBJECTIVE: To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. METHODS: The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. RESULTS: Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. CONCLUSIONS: Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.