Cocaine-stimulated endothelin-1 release is decreased by angiotensin-converting enzyme inhibitors in cultured endothelial cells.

OBJECTIVE: The primary aim was to determine the action of pathophysiologically relevant cocaine concentrations (10(-7)-10(-5) M) on endothelin-1 (ET-1) release from cultured endothelial cells under various cellular conditions. Further aims were to evaluate the effect of angiotensin-converting enzyme inhibitors on cocaine-treated endothelial cells, to assess their potential for inhibition of ET-1-stimulated release. METHODS: Endothelin-1 release into the media was evaluated by radioimmunoassay under basal conditions and after 24 h treatment of endothelial cells with cocaine hydrochloride (HCl), or cocaine HCl and ACE inhibitors, captopril and lisinopril. The effect of serum and plasma under these conditions was also investigated. RESULTS: Cocaine HCl stimulated ET-1 release in a dose response fashion that was independent of plasma or serum factors. Furthermore, cocaine-stimulated ET-1 release was inhibited by administration of angiotensin-converting enzyme inhibitors captopril and lisinopril. CONCLUSIONS: These findings suggest that cocaine can directly stimulate endothelial cells to release ET-1 and that the observed increase is independent of serum or plasma factors. Furthermore, cocaine-stimulated endothelin-1 release appears to be mediated at least in part by the angiotensin system. These observations provide a framework for understanding the cellular mechanisms involved in cocaine-induced vasoconstriction.

Divergent and invariant HLA class II beta chain isoelectric points.

Class II molecules were isolated from consanguineous HTCs (DR1-DRw8) by sequential immunoprecipitation with the monoclonal antibodies 7.3.19.1 (anti-DRw52-like), B8.11.2 (anti-DR backbone), and 7.5.10.1 (anti-HLA class II backbone). Depending upon the DR-serotype of the cell line used, two or three class II antigen families, distinct in molecular weight, could be isolated (see Hum Immunol 9:221, 1984). Immunoprecipitated class II molecules were treated with NaNase and then analyzed on 1D-IEF gels. Each HLA class II antigen family contained two alpha chains conserved in pI. Furthermore, the various haplotypes show distinct electrophoretic beta chain patterns. The number of beta chain charge configurations detected varies from 2 to 5, depending upon the antigen family or haplotype studied. Some of these chains have a pI which is specific for a given class II serotype whereas other beta chain pIs are invariant and shared among more antigen families or haplotypes.

Polymorphisms within the HLA-DRw6 haplotype. III. DQ alpha and DQ beta polymorphism associated with HLA-D.

We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQ alpha and DQ beta polymorphism. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQ alpha and beta chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.

Hyperoxia stimulates endothelin-1 secretion from endothelial cells; modulation by captopril and nifedipine.

PURPOSE: Retinopathy of prematurity (ROP) is a vasoproliferative condition that can result in severe visual impairment and blindness in preterm babies. Two conditions seen very early in radioimmunoassay (ROP) are vasoconstriction and vaso-obliteration. A potent vasoconstrictor secreted by endothelial cells is endothelin-1 (ET-1). Premature birth results in a relative systemic hyperoxia, compared to the in utero oxygen milieu. We tested the hypothesis that hyperoxia increases ET-1 expression as a possible mechanism for vasoconstriction in the retinal vasculature. METHODS: Bovine retinal endothelial cells and adrenal capillary endothelial cells were isolated and maintained in culture. Cells were exposed to control or hyperoxic culture conditions for 24 h, with and without addition of captopril and nifedipine. Media was collected and assayed for ET-1 by ROP. In addition, cell counts and secreted LDH assays were performed. RESULTS: Conditioned media from cultured bovine retinal and adrenal endothelial cells exposed to hyperoxic culture conditions for 24 h were found to have higher levels of ET-1 than conditioned media from normoxic control cells. Captopril (10(-6) M and 10(-4) M) and nifedipine (10(-6) M and 10(-4) M) inhibited ET-1 release from hyperoxia-exposed endothelial cells. Under normoxic conditions, ET-1 release was inhibited by 10(-4) M captopril or 10(-4) M nifedipine. CONCLUSIONS: These results demonstrate that (1) hyperoxia stimulates in vitro ET-1 secretion in bovine retinal and adrenal capillary endothelial cells, and (2) captopril and nifedipine downregulate ET-1 secretion under normoxic and hyperoxic culture conditions, in a dose-dependent fashion. We speculate that ET-1 may be involved in retinal vessel vasoconstriction seen early in the development of ROP. Further, ACE inhibitors and calcium-channel blocking agents, such as captopril and nifedipine, may provide an avenue for blocking vasoconstriction in ROP.

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

Antimalarial activity of cyclosporins A, C and D.

Cyclosporin A (CsA) is an immunosuppressive drug widely used in organ transplants. It is also accumulated by the erythrocyte, a site that accommodates one of the stages of malaria parasite. We observe that CsA and its less potent immunosuppressive analogues CsC and CsD were as effective as chloroquine in inhibiting P. berghei malaria parasite development in vivo (when administered orally) and P. falciparum parasite in vitro. They were, however, not inhibitory to the liver stages and the gametocytes. In vivo the minimum effective dose was 10 mg/Kg administered on two consecutive days whereas, in vitro CsA and its analogues inhibited parasite development at concentrations of 10 micrograms/ml and above.

Perceptions, health care seeking behaviour and implementation of a tuberculosis control programme in Lambaréné, Gabon.

SETTING: Lambaréné, Gabon. OBJECTIVES: To describe patient perceptions of tuberculosis (TB) and to determine factors that influence health care seeking behaviour to gain insight into the management of multidrug-resistant TB. DESIGN: Participant observation, in-depth semi-structured interviews and focus group discussions were conducted with 30 TB patients, 36 relatives, 11 health care providers and 18 traditional/spiritual healers. Recruitment of patients was linked to the PanEpi study and took place at the Albert Schweitzer Hospital, the General Hospital and the TB-HIV (human immunodeficiency virus) clinic. RESULTS: Patients generally described TB as a natural and/or magical disease. The majority of the patients combined treatment at the hospital with (herbal) self-treatment and traditional/spiritual healing. Despite the free availability of anti-tuberculosis treatment in principle, patient adherence was problematic, hindering effective TB control. Most patients delayed or defaulted from treatment due to financial constraints, stigmatisation, ignorance about treatment, change of health care service or use of non-prescribed antibiotics. The situation was occasionally complicated by drug stockouts. CONCLUSION: There is an urgent need to bridge the gap between patients and the hospital by avoiding drug shortages, intensifying culturally sensitive TB health education, embedding TB care into the cultural context and enhancing cooperation between hospitals, patients, traditional healers and communities.

Human endometrial endothelial cells: isolation, characterization, and inflammatory-mediated expression of tissue factor and type 1 plasminogen activator inhibitor.

Binding of Ulex europaeus lectin to microvessels was used to isolate endothelial cells from cycling human endometrium. Cultured human endometrial endothelial cells (HEECs) exhibited endothelial cell-specific characteristics such as tube formation on a basement membrane matrix and sequestration of acetylated low-density lipoprotein. Markers for potentially contaminating epithelial, stromal, smooth muscle, and bone marrow-derived cells were not detected in the HEEC cultures. Basal and proinflammatory-stimulated immunostaining profiles for endothelial cell-specific adhesion markers, as exemplified by Von Willebrand's factor and E-selectin, were similar for cultured HEECs and human umbilical venous cord endothelial cells (HUVECs). However, HUVECs expressed several extracellular matrix proteins that were absent from cultured HEECs. In the latter, the protein kinase C agonist phorbol myristate acetate transiently enhanced tissue factor (TF) mRNA levels and elicited a more prolonged elevation in TF protein levels, but did not affect plasminogen activator inhibitor-1 (PAI-1) mRNA and protein levels. Inappropriate expression of TF, which initiates hemostasis by generating thrombin, and of PAI-1, which regulates hemostasis by acting as the primary inhibitor of fibrinolysis, can each lead to thrombosis. The differential regulation of TF and PAI-1 expression revealed in the current study emphasizes the importance of using HEECs to evaluate mechanisms regulating the hemostatic/thrombotic balance in human endometrium.

The major histocompatibility complex of rhesus monkeys, RhLA: XV. Chemical characterizations of DR locus antigens and antigen 48.

Immunoprecipitation studies of the rhesus monkey major histocompatibility system have shown that the RhLA-DR locus codes for class II antigens with molecular features that are homologous to the class II antigens coded for by the human HLA-DR locus. The product of another alloantigenic RhLA-linked locus of the rhesus monkey, called '48', is provisionally characterized as a class I system.

Polymorphisms within the HLA-DRw6 haplotype. II. Protein charge heterogeneity reflects MLC subtyping of HLA-DRw6 homozygous cells.

We have analyzed the HLA class II gene products from HLA-DRw6 homozygous cells. Epstein-Barr virus-transformed B-cell lines were internally labeled with [35S]-methionine. An NP-40 lysate of the cells was subjected to immunoprecipitation, first with a DRw52-like-specific monoclonal antibody and subsequently with a DR-specific framework antibody. The DR region-encoded gene products were analyzed by one-dimensional gel isoelectric focusing and two-dimensional gel electrophoresis. It is shown that DRw6 homozygous cell lines contain at least two nonallelic DR beta chains, one carrying a DRw52 determinant and one DRw52-negative population. Both chains appear to be polymorphic between the cellularly defined subtypes of DRw6. The determinant responsible for the differential mixed lymphocyte culture reactivity of Dw18 and Dw19 cells resides on the DRw52-positive population, whereas the Dw6-Dw9 differences are attributed to determinants on both populations of DR light chains. The Dw16-derived DRw52+ chain much resembles the Dw18 DRw52+ light chain whereas there is a clear-cut difference between these two subtypes in the DRw52- population. We conclude that, for DRw6 homozygous cells, the cellularly recognized D determinants are probably located on DR-encoded molecules, both DRw52+ and DRw52-, and that charge shift of these chains is at least partly responsible for differential recognition of these cells in mixed lymphocyte cultures.

Endothelin-1 vasoactive responses in lambs with pulmonary hypertension and increased pulmonary blood flow.

Increased concentrations of endothelin-1 (ET-1) are found in children with congenital heart diseases that produce increased pulmonary blood flow and pulmonary hypertension, but the role of ET-1 in the pathophysiology of pulmonary hypertension is unclear. Therefore, we investigated ET-1-induced vasoactive responses and ET-1 concentrations in an animal model of pulmonary hypertension and increased pulmonary blood flow. Vascular shunts were placed between the ascending aorta and main pulmonary artery in seven late-gestation fetal sheep. Four weeks after spontaneous delivery, ET-1 increased pulmonary vascular resistance by 29.7 +/- 34.4% (P < 0.05), the ETb-receptor agonist [Ala1,3,11,15]ET-1 (4AlaET-1) had no effect, and the ETa-receptor antagonist cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp) (BQ-123) decreased pulmonary vascular resistance by -16.0 +/- 5.6% (P < 0.05). In contrast, in six control lambs with a similar degree of pulmonary hypertension induced by U-46619, ET-1 and 4AlaET-1 decreased pulmonary vascular resistance by 24.8 +/- 17.6, and 20.0 +/- 13.8%, respectively (P < 0.05). In addition, systemic arterial concentrations of immunoreactive ET-1 were elevated in lambs with pulmonary hypertension (29.2 +/- 9.6 vs. 15.2 +/- 10.7 pg/ml, P < 0.05). Pulmonary hypertension and increased pulmonary blood flow alters the response of ET-1 from pulmonary vasodilation to vasoconstriction. These altered responses suggest a role for ET-1 and its receptors in the pathogenesis of pulmonary hypertension secondary to increased pulmonary blood flow.

Expression of angiopoietin-2 by human endometrial endothelial cells: regulation by hypoxia and inflammation.

The functional endometrial layer receives the implanting blastocyst, but is sloughed off during menstruation. Angiogenesis regulates growth and repair of cycling human endometrium. While vascular endothelial growth factor initiates angiogenesis, the angiopoietins (Angs) acting via the Tie2 receptor, are key regulators of subsequent angiogenic steps. This study is the first to localize Ang-2 and Tie2 in human endometrium and to study Ang-2 regulation in cultured human endometrial endothelial cells (HEECs). Immunohistochemistry revealed that expression of Ang-2 and Tie2 was absent from the glands, low in stromal cells, and intense in the endothelial cells. In contrast, only weak expression of Ang-1 was detected. The phase of the menstrual cycle did not appear to affect the expression of Ang-2 or Tie2. In vitro studies were carried out utilizing isolated HEECs, the most relevant model for endometrial microvascular biology studies. Both hypoxia and phorbol-myristate-acetate enhanced Ang-2 mRNA levels in HEECs. These results suggest that Ang-2 plays a role in endometrial pathologies complicated by impaired blood flow and inflammation.

Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1.

Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ET(A) receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 +/- 42.2% (P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1alpha, and ET(A) and ET(B) receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% (P < 0.05) in control lambs but was unchanged (-5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ET(A) receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.

Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation.

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.