High prevalence of adrenal insufficiency in patients with sickle cell disease: results from a community hospital in the U.S.

OBJECTIVE: Despite the low prevalence (0.008%) of adrenal insufficiency (AI) in the general population, this disorder was recently diagnosed in a substantial number of sickle cell disease (SCD) patients at our hospital. The main objective of this study was to assess the prevalence of AI in SCD patients. METHODS: All adult patients admitted to the Department of Medicine at Interfaith Medical Center from October 2010 to November 2011 were eligible for this retrospective study. Medical records of adult SCD patients hospitalized for painful crisis and who had undergone cosyntropin testing were reviewed. Adult non-SCD patients hospitalized for painful crisis and who had undergone cosyntropin testing served as controls. The result of the cosyntropin test was the primary outcome. The prevalence of positive cosyntropin tests was compared between the 2 groups by using Student's t-test, and odds ratios. RESULTS: 62 adult SCD patients were enrolled in the study. 15 underwent cosyntropin testing and 12 (19.4%) of these patients were found to have AI. AI was also diagnosed in 1 of 1,340 non-SCD patients. The odds ratio for AI in SCD to non-SCD patients [(12/62)/(1,340)] was 259. The odds ratio for the prevalence of AI in SCD patients in our study (19.4%) vs. the general population (approximately 0.008%) was 2,375. CONCLUSION: AI occurred in 19.4% of SCD patients included in this study. These patients thus have a 2,375-fold higher risk of developing AI than the general population, and a 259-fold greater risk of developing AI than do hospitalized non-SCD patients.

Gastric marginal zone B cell lymphoma of the duodenum.

Small bowel lymphomas of the extranodal type occur in the young and are characteristically associated with malabsorption syndrome. We present the case of an elderly in whom there was no malabsorption and the duodenal tumor was a gastric type marginal zone B cell lymphoma also known as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. A 73-year-old woman presented to the emergency room with 2 weeks of general weakness, recurrent vomiting containing food particles and abdominal distension. She had been diagnosed with diabetic gastroparesis 4 years prior. CT of the abdomen and pelvis was suggestive of gastric outlet obstruction but no evidence of pancreatic or duodenal mass. Endoscopy and biopsy of the tumor obstructing the distal first part of the duodenum confirmed a gastric marginal MALT lymphoma. The patient's symptoms improved with radiotherapy. Gastric MALT lymphoma, an extranodal lymphoma primarily described in the stomach, can also present in the small bowel and is not associated with malabsorption.

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

Thrombotic thrombocytopenic purpura and sporadic hemolytic-uremic syndrome plasmas induce apoptosis in restricted lineages of human microvascular endothelial cells.

Thrombotic thrombocytopenic purpura (TTP) and sporadic hemolytic-uremic syndrome (HUS) are thrombotic microangiopathies that occur in the absence of an inflammatory response. Ultrastructural features of tissues involved in TTP/sporadic HUS suggest an apoptotic process. Consistent with these findings, we observed that TTP plasmas induce apoptosis in primary human endothelial cells (EC) of dermal microvascular but not umbilical vein origin (Laurence et al, Blood 87:3245, 1996). We now document the ability of plasmas from both TTP and sporadic HUS patients, but not from a patient with childhood/diarrhea-associated HUS, to induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) in restricted lineages of microvascular EC. EC of small vessel dermal, renal, and cerebral origin were susceptible to induction of Fas and an apoptotic cell death. In contrast, microvascular EC of pulmonary and hepatic origin, as well as EC of a large vessel, coronary artery, were resistant to both processes. This dichotomy parallels the in vivo pathology of TTP/sporadic HUS, with notable sparing of the pulmonary and hepatic microvasculature. Apoptotic EC also had some features of a procoagulant phenotype, including depressed production of prostaglandin I2 (prostacyclin). These phenomena support the pathophysiologic significance of microvascular EC apoptosis in TTP, extend it to a related disorder (sporadic HUS), and suggest consideration of apoptosis inhibitors in the experimental therapeutics of these syndromes.

Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium.

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.

Hypoglycemia stimulates thrombin-induced PGI2 production by cultured human umbilical vein endothelial cells.

The effect of a low concentration of glucose on prostacyclin (PGI2) production by human umbilical vein endothelial cells in culture (HUVEC) was examined. When HUVEC were cultured for 24 h in a medium containing various concentrations of glucose (0.26, 0.63, or 1.29 mg/ml), thrombin-induced PGI2 production was increased in the cells exposed to low concentrations of glucose; the increase was inversely proportional to the glucose concentration and was seen at all concentrations of thrombin tested. Thus, hypoglycemia may increase the production of PGI2 by endothelial cells in vivo, leading to a compensatory vasodilation with an increase in blood flow to increase the delivery of glucose.

Identification of a high molecular weight endothelial cell surface glycoprotein, endoGlyx-1, in normal and tumor blood vessels.

BACKGROUND: The vascular endothelium participates in diverse physiologic and pathologic processes, ranging from blood clotting to leukocyte trafficking and tumor metastasis. Many of these functions are mediated by endothelial surface molecules, and monoclonal antibodies (mAb) have been used extensively to define endothelial cell surface structures in normal and lesional tissues. EXPERIMENTAL DESIGN: MAbs raised against human umbilical vein endothelial cells were tested on a panel of cultured cell types representing diverse cell lineages. In vivo antigen expression was examined through immunohistochemical tests with normal and lesional tissues. Immunochemical methods were employed to characterize mAb-defined antigens in endothelial cell extracts. RESULTS: The newly-derived mAbH572 reacts with a high molecular weight glycoprotein complex of cultured endothelial cells comprised of disulfide-bonded subunits of molecular weight 190,000, 140,000, 125,000, and 110,000. This antigen, designated endoGlyx-1, is expressed on the surface of cultured endothelium, with no changes in expression after cytokine-stimulation. Immunohistochemical assays detect endoGlyx-1 in endothelial cells of large, medium-sized, and small blood vessels, including capillaries, in all human organs tested; only hepatic and splenic sinusoids lack the molecule. EndoGlyx-1 is not found in any of the normal fetal and adult nonendothelial cell types tested. In a study of > 100 tumors, endoGlyx-1 immunostaining was consistently found in tumor capillaries, including "hot spots" of neoangiogenesis in certain tumors. EndoGlyx-1 is also expressed in hemangiomas and angiosarcomas. CONCLUSIONS: The highly restricted expression of endoGlyx-1 in normal and tumor blood vessels suggests a role for this molecule unique to endothelial cell physiology. In fact, endoGlyx-1 appears to be the only endothelial lineage-specific cell surface glycoprotein identified to date. The availability of endoGlyx-1 antibodies may aid in the characterization and quantification of the angiogenic phase of solid tumors and facilitate the search for endothelial precursors in bone marrow or peripheral blood.

Schistosoma mansoni egg attachment to cultured human umbilical vein endothelial cells: an in vitro model of an early step of parasite egg excretion.

To model the attachment of Schistosoma mansoni eggs to the endothelium of the mesenteric vasculature, the interaction between S. mansoni eggs and cultured human umbilical vein endothelial cells (HUVEC) in vitro was investigated. S. mansoni eggs rapidly attached to monolayers of both HUVEC and bovine aortic endothelial cells but more slowly to monolayers of cultured fibroblasts and smooth muscle cells. While both native and glutaraldehyde-fixed eggs attached equally well to HUVEC, eggs attached only to live, metabolically active HUVEC. Attachment was enhanced by both serum and plasma factors. In addition, platelet release products increased egg attachment by 75%. Preincubation of S. mansoni eggs with soluble egg antigens promoted attachment; in contrast, preincubation of HUVEC with the antigens inhibited attachment. These results suggest that interaction of S. mansoni eggs with HUVEC is an active process that can be modulated by molecules secreted by the egg and by platelets during egg extravasation.

Comparison of the potency of various serotypes of E. coli lipopolysaccharides in stimulating PGI2 production and suppressing ACE activity in cultured human umbilical vein endothelial cells.

We investigated the potency of various serotypes of lipopolysaccharides (LPS) by examining LPS-induced stimulation of PGI2 production and suppression of ACE activity in cultured human umbilical vein endothelial cells (HUVEC). HUVEC which had been incubated with E. coli 055:B5 and 0111:B4 for 24 h produced more prostacyclin (PGI2) in response to thrombin than HUVEC incubated with E. coli 026:B6. Also, angiotensin converting enzyme activity (ACE) in cell lysates of HUVEC incubated for 24 h with 055:B5 or 0111:B4 was suppressed significantly compared to control HUVEC or HUVEC incubated with 026:B6. From these experimental results, E. coli 055:B5 and 0111:B4 appear to be more potent than 026:B6. It is concluded that this difference in potency among various serotypes of LPS should be taken into account when experiments are designed to examine the effect of LPS on endothelial cell function.

Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer.

Cell surface antigens of transformed cells are the traditional targets for antibody-guided detection and therapy of solid neoplasms. Alternative targets may be found in the tumor stroma, which contains newly formed blood vessels, reactive fibroblasts, and extracellular matrix proteins. The F19 cell surface glycoprotein of reactive fibroblasts is a prototypic stromal antigen since it is found in the stroma of > 90% of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors. In the present study, we define an additional tumor stromal antigen, FB5, that is selectively expressed in vascular endothelial cells of malignant tumors. Immunohistochemical analysis of 128 tumors identified FB5 in endothelial cells in 67% of the samples (including 41 of 61 sarcomas, 26 of 37 carcinomas, and 18 of 25 neuroectodermal tumors) whereas normal blood vessels and other adult tissues tested lacked FB5 expression. In vitro studies showed that FB5 is a M(r) 165,000 cell surface glycoprotein, comprised of a M(r) 95,000 core polypeptide and highly sialyated O-linked oligosaccharides but few if any N-linked sugars, and that the FB5 gene is located on chromosome 11q13-qter. The restricted tissue distribution of the FB5 protein, which we refer to as endosialin, suggests strategies for tumor imaging and therapy that are aimed primarily at the tumor vasculature.

Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor.

Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the beta/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect beta/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the beta/A4 amyloid protein precursor via the secretory cleavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.

The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.

Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to cultured human endothelial cells.

Tumor necrosis factor (TNF), a potent inflammatory mediator secreted by monocytes during inflammation, was shown to significantly increase the adherence of Staphylococcus aureus to cultured human umbilical vein endothelial cells in vitro. The stimulatory effect of TNF was dose dependent and was bimodal with respect to time; bacterial adhesion peaked after 4 and 16 h of stimulation with recombinant human TNF-alpha. The ability of TNF-alpha to augment staphylococcal adherence to endothelial cells was contingent upon the presence of plasma factors. Thus, the complex interaction among cytokines (such as TNF), plasma factor(s), and the endothelium serves to modulate bacterial adherence to endothelial cells.

A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells.

Cleavage by thrombin of the platelet thrombin receptor exposes a new N-terminal segment SFLLRNPNDKYEPF (SFLL) which acts as a tethered ligand. The free peptide activates platelets and induces platelet aggregation. We now show that SFLL can also activate human umbilical vein endothelial cells (HUVEC) and induce rises in both cytosolic free calcium ([Ca2+]i) and prostacyclin (PGI2) production. These responses were time- and concentration-dependent and were similar to those for native thrombin except that they were not blocked by hirudin. Initial activation of HUVEC with thrombin desensitized the subsequent response to SFLL for both rises in [Ca2+]i and PGI2 production. Thus, SFLL alone can activate HUVEC and elevate [Ca2+]i and induce PGI2 production suggesting that the thrombin receptors on platelet and endothelial cells are functionally and structurally similar.