Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.     

Knockout-transgenic mouse model of sickle cell disease

When transgenic mice that expressed human sickle hemoglobin were mated with mice having knockout mutations of the mouse alpha- and beta-globin genes, animals were produced that synthesized only human hemoglobin in adult red blood cells. Similar to many human patients with sickle cell disease, the mice developed a severe hemolytic anemia and extensive organ pathology. Numerous sickled erythrocytes were observed in peripheral blood. Although chronically anemic, most animals survived for 2 to 9 months and were fertile. Drug and genetic therapies can now be tested in this mouse model of sickle cell disease.     

Endothelin in human nasal mucosa

Endothelin (ET), a potent vasoconstrictor and bronchoconstrictor peptide synthesized by endothelial and epithelial cells, was examined for its potential functions in human inferior turbinate nasal mucosal tissue by four techniques: (1) immunoreactive ET was localized in the mucosa by immunohistochemistry; (2) receptors for ET were identified by autoradiography employing [125I]ET; (3) ET-1 mRNA was localized by in situ hybridization; and (4) the secretory functions of ET were examined by the release of mucous and serous cell products after the addition of ET to human nasal turbinates in short-term cultures. Specific ET-1-immunoreactive material was found most extensively in small muscular arteries and in serous cells in submucosal glands. ET-1 was also found to a lower extent in the walls of venous sinusoids. [125I]ET-1 binding sites were localized by autoradiography to submucosal glands and to venous sinusoids and small muscular arterioles. mRNA for ET-1 was found most extensively in the venous sinusoids and to a lesser extent in small muscular arteries. In mucosal explant cultures, ET-1 and ET-2 stimulated lactoferrin and mucous glycoprotein release from serous and mucous cells, but ET-3 was inactive. The observations indicate that in the human nasal mucosa, ET is present in the vascular endothelium and the serous cells in submucosal glands and acts on glandular ET receptors to induce both serous and mucous cell secretion. It is also likely that ET plays a role in the regulation of vasomotor tone.     

Differences in the yields of dicentrics and reciprocal translocations observed in the chromosomes of irradiated human skin fibroblasts and blood lymphocytes from the same healthy individuals

The modulatory role of locally produced cyclooxygenase products and endothelium-derived nitric oxide in controlling vascular tone was investigated in bovine intra-mammary artery. Vascular reactivity initiated by vasoactive compounds, endothelin-1 (ET-1), bradykinin (BK), and substance P (SP) was measured isometrically in an isolated tissue bath. The effects of a cyclooxygenase inhibitor, indomethacin (10(-5) M) and an inhibitor of nitric oxide production, N omega-Nitro L-Arginine (L-NNA: 3 x 10(-4) M) were determined during agonist-mediated responses. Indomethacin alone markedly enhanced vascular contraction produced by ET-1, while L-NNA did not. Inhibition of endothelium-derived nitric oxide synthesis by L-NNA, however, significantly attenuated BK- and SP-induced vascular relaxations, whereas indomethacin had slight influence. The potentiation between indomethacin and L-NNA in regulating vasomotor tone was not observed in this vascular bed. Thus, it appeared that both the cyclooxygenase and endothelium-derived nitric oxide pathways participated in modifying vascular reactivity. Domination of one pathway over the other depended upon the agonist used to stimulate vascular tissue.     

Circulating activated endothelial cells in sickle cell anemia

BACKGROUND: The vascular wall participates in the pathogenesis of sickle cell disease. To determine whether the endothelium is activated in this disease, we studied the number, origin, and surface phenotype of circulating endothelial cells in patients with sickle cell anemia. METHODS: We used immunohistochemical examination of buffy-coat smears to enumerate circulating endothelial cells, and we evaluated the surface phenotype by applying preparations of circulating endothelial cells. An immunofluorescence microscopy panel of antibodies was used, including a specific anti-endothelial-cell antibody, P1H12. RESULTS: Mean (+/-SD) numbers of circulating endothelial cells in normal blood donors, patients with sickle cell trait, and patients with hemolytic anemias not due to hemoglobin S were 2.6+/-1.6, 3.0+/-2.6, and 2.0+/-0.8 per milliliter of whole blood, respectively. Patients with sickle cell anemia who presented with acute painful episodes had 22.8+/-18.2 circulating endothelial cells per milliliter of blood (P<0.001 for the comparison with normal donors), and patients with no such events within one month before or after blood sampling had 13.2+/-11.8 circulating endothelial cells per milliliter of blood (P=0.002 for the comparison with normal donors and P=0.019 for the comparison with patients with acute events). Serial observations of three patients showed a tendency toward higher levels of circulating endothelial cells at the onset of acute painful crises. The average viability of circulating endothelial cells was 66+/-30 percent. In patients with sickle cell anemia, regardless of clinical status, the circulating endothelial cells were predominantly microvascular in origin (CD36-positive), and most of the cells expressed four markers of endothelial-cell activation: intercellular adhesion molecule 1, vascular-cell adhesion molecule 1, E-selectin, and P-selectin. CONCLUSIONS: Our studies suggest that the vascular endothelium is activated in patients with sickle cell anemia, regardless of the patients' clinical status. Adhesion proteins on activated endothelial cells may have a role in the vascular pathology of sickle cell disease.     

The role of CT and MR in imaging the complications of sickle cell disease

Sickle cell disease is the most common inherited haemoglobinopathy described. Complications of sickle cell disease (SCD) are due to chronic haemolysis of fragile red cells or secondary to vascular occlusion by sickled red cells with subsequent tissue infarction. Traditionally plain film radiography has been the mainstay in the assessment of patients with SCD, but increasingly magnetic resonance (MR) imaging and computed tomography (CT) are being used. In this review the imaging features of a range of complications of SCD are demonstrated with particular emphasis on CT and MR.     

Endothelial cells exposed to erythrocytes under shear stress: an in vitro study

After injury and vascular replacement, endothelial cell recovery is limited and could lead to thrombosis. Seeding small diameter vascular prosthesis with endothelial cells has been proposed to fulfil cell lining and improve surface hemocompatibility. However, detachment of seeded cells occurs following implantation. Previous in vitro studies have looked at the fluid shear stress as a major cause of cell detachment. To our knowledge, the role of erythrocyte collisions has not been investigated. The present in vitro study aims at investigating whether endothelial cell adhesion depends on (i) the presence of erythrocytes in flow and (ii) the latent culture period (1, 24 and 48 h) between seeding and exposure to flow. Endothelial cells were exposed to culture media containing different erythrocyte concentrations using a steady laminar flow of 1350 ml min(-1) in a parallel plate flow chamber. Endothelial cell morphology in dynamic conditions was quantified and compared to that in static conditions. The projected area of cells were mostly found smaller under dynamic than static conditions, particularly at a wall shear stress of 23 dyn cm(-2). Cells from the 1 h latent culture period were oriented parallel to the flow axis and were more elongated than under static conditions. Conversely, endothelial cell shape was slightly modified when either the latent period or the wall shear stress was increased. Disparate orientation was observed on confluent endothelial cells (24-48 h latent period) exposed to shear stress with or without erythrocytes. Increasing fluid viscous forces due to erythrocytes play a critical role on the behaviour of freshly seeded endothelial cells upon exposure to blood flow.     

Alternative splicing of CYP2D mRNA in human breast tissue

Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction-specifically, deregulation of endothelin-1 (ET-1) secretion-contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge-Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.     

Long-term evaluation with DMSA-Tc 99m scintigraphy of renal parenchymal involvement in children after shockwave extracorporeal lithotripsy

BACKGROUND: Endothelin 1 (ET-1), a vasoconstrictor peptide, has been implicated as a tumour growth stimulator and an angiogenesis factor. METHODS: To assess the involvement of ET-1 in colorectal cancer, immunoelectron microscopy for ET-1 was performed in colorectal liver metastases and normal liver (n = 6). ET-1 plasma levels were measured by radioimmunoassay in patients with colorectal cancer, with (n = 18) and without (n = 12) liver metastases, and in controls (n = 22). RESULTS: In normal liver, ET-1 was present in endothelial cells; in tumour, it was observed in endothelial cells, tumour cells and myofibroblasts. Mean(s.d.) plasma ET-1 levels were 2.75 (1.37) pg/ml in controls, 4.53(1.61) pg/ml in patients with colorectal liver metastases (P = 0.001) and 3.92(1.32) pg/ml in patients without metastases (P = 0.02). CONCLUSION: ET-1 was present in various cell types within colorectal liver metastases and raised levels were found in the plasma of patients with colorectal cancer. ET-1 may not only modulate tumour vascular tone but also act on tumour growth and angiogenesis, both locally and systemically.     

Hypomagnesaemia in postoperative patients: an important contributing factor in postoperative mortality

Nine patients with sickle cell disease (SCD) were operated upon at our hospital for acute appendicitis, comprising only 0.43% of the total appendicectomies performed at our institution. Three appendices were acutely inflamed and six (66.7%) were perforated. Histologic evaluation of the six perforated specimens revealed congestion and haemorrhage by sickled erythrocytes (RBCs) in addition to acute transmural inflammatory cell infiltrates. The mucosa was extensively ulcerated, with haemorrhage both within the lumen and in the appendiceal wall. The blood vessels were dilated and packed with sickled RBCs. Two of the three acutely inflamed appendices showed features of acute transmural appendicitis, with marked congestion and haemorrhage by sickled RBCs. The third did not show any acute inflammatory cell infiltrate, however, the mucosa was partly ulcerated with both mucosal and intraluminal haemorrhage. These findings suggest that acute appendicitis is different in patients with SCD: while it is not common, when it does develop it has a rapid course with a high incidence of perforation due to blockage of appendiceal vessels by sickled RBCs, leading to transmural necrosis.     

Endothelin-1 levels in ischaemia, reperfusion, and haemorrhagic shock in the canine infrarenal aortic revascularisation model

Endothelin-1 (ET-1) is a potent vasoconstrictive polypeptide produced from vascular endothelial cells. The effects of ischaemia, reperfusion, and exsanguination on plasma ET-1 levels were studied and compared in the mongrel dog after infrarenal aortic cross clamping. Ischaemia produced a trend toward increased ET-1 serum levels (p < 0.07 with Bonferroni correction) that did not reach significance. Plasma ET-1 levels were significantly increased during reperfusion and even further elevations were found following exsanguination. We found a 2-3 fold increase in ET-1 levels following reperfusion (Initial 3.19 +/- 0.27 pg/ml vs. Reperfusion maximum 6.32 +/- 0.72 pg/ml, Bonferroni p < 0.01). Haemorrhagic shock was associated with a 3-4 fold increase in ET-1 levels (Initial 3.19 +/- 0.27 pg/ml vs. Exsanguination maximum 8.37 +/- 0.97 pg/ml Bonferroni p < 0.001). These data reveal that ET-1 is released during reperfusion and exsanguination and may mediate remote vascular events associated with infrarenal aortic cross clamping and acute blood loss.     

Increased immunoreactive endothelin-1 in human transplant coronary artery disease

BACKGROUND: The pathogenesis of transplant coronary artery disease (TCAD) is unknown, but it is thought to derive from an interaction between immune and nonimmune factors, leading to smooth muscle cell proliferation and accumulation in the expanded neointima. Endothelin-1 (ET-1), a potent vasoconstrictor with mitogenic properties for vascular smooth muscle cells, has recently been demonstrated in native vessel atherosclerosis. The present study used immunohistochemistry to investigate the role of ET-1 in TCAD. METHODS AND RESULTS: ET-1 immunoreactivity and cellular localization were assessed in human coronary arteries with TCAD (n = 13) and in normal coronary arteries (n = 10) with single- and double-label immunohistochemistry. The intensity of immunostaining was determined by a semiquantitative method. Diffuse and intense ET-1 immunoreactivity was found in 11 of 13 patients with TCAD (85%), mainly in myointimal cells and, in lesser amounts, in macrophages and endothelial cells. In contrast, normal coronary arteries had only faint immunostaining localized to the endothelial layer. Mean semiquantitative grade was significantly higher in TCAD than in normal arteries (1.8 versus 0.7; P < .05). ET-1 was more frequently present in lipid-rich, atheromatous lesions than in lipid-poor, proliferative ones. Intimal neovessels consistently immunostained for ET-1. CONCLUSIONS: Immunoreactivity for ET-1 is significantly increased in TCAD, possibly as a result of stimulatory cytokines and growth factors that are upregulated in the posttransplant state. The results suggest a role for this mitogenic peptide in the pathogenesis of graft arteriosclerosis.     

Stellate neurones innervating the rat heart express N, L and P/Q calcium channels

BACKGROUND: Distension of the saphenous vein before and after coronary artery bypass grafting results in damage to mechanisms that regulate vascular tone. We have investigated the relationship between the magnitude of distending pressure and the degree of structural, biochemical and functional damage to the vessel wall. METHODS: Vessel segments that had been distended to either 100 or 300 mmHg were set up in isolated organ baths and the function of the smooth muscle and endothelial cells examined. All segments examined were then fixed for assessment of structural damage by scanning electron microscopy and for immunocytochemical localisation of endothelial nitric oxide synthase. RESULTS: Segments of saphenous vein distended to 100 mmHg retained their responsiveness to KCl (90 mmol/l) and phenylephrine (10(-6) mol/l), but those pressurised to 300 mmHg had significantly reduced responses to both agents. There was also a significant reduction in response to the endothelium-dependent dilators, acetylcholine (10(-10)-10(-6) mol/l) and bradykinin (10(-10)-10(-6) mol/l) in those segments distended to 300 mmHg. Quantitative studies of structural endothelial damage showed a significant loss of endothelium at 300 mmHg distension pressure. Remaining endothelial cells retained strong positive staining for endothelial nitric oxide synthase. By electron microscopic examination, those vessels distended to 100 mmHg showed lifting and rounding of individual cells, whereas segments distended to 300 mmHg revealed major areas of denuded endothelium. CONCLUSIONS: Distension of saphenous veins to pressures equivalent to those in the systemic circulation result in structural and biochemical changes in the endothelium that are not paralleled by immediate functional vasomotor changes.     

Ultrastructure of sickling and unsickling in time-lapse studies

The denser subpopulation of erythrocytes from patients with sickle cell anaemia was deoxygenated to a pO2 of 4.7 kPa or reoxygenated to a pO2 of 12 kPa with a continuous-flow apparatus. Samples were collected into modified Karnovsky's fixative at intervals between 0.5 and 15 S. .The earliest event after deoxygenation was aggreagation of haemoglobin followed by the formation of fibres of 160-200 A diameter. The polymers were always randomly distributed in a loose network. A highly ordered, close packing of fibres characteristic of the nematic liquid crystal was not achieved within 15 S. Depolymerization involved a shortening of fibres followed by aggregation similar to that observed early in the polymerization process and prior to the return to the unperturbed state. Irreversibly sickled cells were the first to demonstrate polymers following deoxygenation and that last to lose polymers after reoxygenation. Polymerization of the haemoglobin preceded the appearnce of the sickled deformity of reversibly sickled cells and, following reoxygenation, the return to the discoid shape lagged behind the disappearance of polymers. These studies, carried out under physiologic conditions, have demonstrated intracellular changes during time intervals that correspond to the normal venous and arterial circulation that may contribute to the pathophysiology of sickling disorders.     

Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.     

Cyclooxygenase products mediate the cutaneous vasodilation induced by endothelin-1 in humans

Intradermal injection of endothelin-1 (ET-1) causes vasoconstriction (pallor) at the injection site, surrounded by a larger area of vasodilation (flare) in humans. Some of the vasomotor responses to ET-1 are thought to be mediated by prostaglandins. In the present study, we investigated the involvement of cyclooxygenase-derived products of arachidonic acid metabolism on the cutaneous vasomotor responses to ET-1. Ten normal subjects (25-44 years) were studied after treatment with either indomethacin (50 mg t.i.d.) or placebo according to a double blind cross-over design. Five doses of ET-1 (5 x 10(-5) to 5 x 10(-1) pmol) were injected intradermally 2 h after the last dose of indomethacin or placebo. Pallor and flare areas measured by planimetry 15 min after the injection were analyzed to evaluate cutaneous vasomotor responses to ET-1. ET-1 induced dose-dependent pallor and flare responses that were significant at the dose of 5 x 10(-3) pmol or greater. Indomethacin did not affect the ET-1-induced pallor but significantly shifted to the right the flare dose-response curve to ET-1. The inhibition of the flare response to 5 x 10(-1) pmol ET-1 was 58.9 +/- 8.5%. These results indicate that the cutaneous vasodilation induced by intradermal injection of ET-1 is mediated by the release of vasodilating cyclooxygenase products.