Margaret Broderius

Cultured human and canine endothelial cells from brain microvessels.

Cultures of endothelial cells (EC) derived from human (autopsy) and canine brain microvessels were characterized with respect to growth, morphology, and biochemical features. The endothelial nature of these cells was confirmed by analyses of angiotensin-converting enzyme activity, Factor VIII-related antigen, and ultrastructure. Human EC required coated substrates and tumor-conditioned medium to achieve rapid growth, and cells derived from human microvessels were morphologically diverse. In contrast, canine EC grew rapidly on plastic substrates and produced colonies of uniform morphology. Morphological variations of EC were associated with the use of heparin-containing medium and with the use of a commercially-prepared basement membrane extract (Matrigel). Lectin histochemistry demonstrated that human EC lack the abundant alpha-galactose residues characteristic of canine EC membranes and organelles and that canine EC lack the alpha-N-acetylgalactosamine residues which are associated with human EC. The lectin Ricinus communis agglutinin I may be useful for distinguishing canine EC from pericytes. Gel electrophoresis of membrane proteins revealed protein bands present in human EC at Mr 210,000 and 37,000-39,500 which were not present in canine EC. These proteins may be related to the presence of junctional complexes in cultures of human EC.

Metallochaperone for Cu,Zn-superoxide dismutase (CCS) protein but not mRNA is higher in organs from copper-deficient mice and rats

Cu,Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats and mice have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. SOD1 interacts with and requires its metallochaperone CCS for donating copper. We produced dietary Cu deficiency in rodents to determine if the reduction in SOD1 was related to the level of its specific metallochaperone CCS. CCS levels determined by immunoblot were 2- to 3-fold higher in liver, heart, kidney, and brain from male Cu-deficient rats and mice under a variety of conditions. CCS was also higher in livers of Cu-deficient dams. Interestingly, CCS levels in brain of Cu-deficient mice were also higher even though SOD1 activity and protein were not altered, suggesting that the rise in CCS is correlated with altered Cu status rather than a direct result of lower SOD1. A DNA probe specific for rat CCS detected a single transcript by Northern blot hybridization with liver RNA. CCS mRNA levels in mouse and rat liver were not altered by dietary treatment. These results suggest a posttranscriptional mechanism for higher CCS protein when Cu is limiting in the cell, perhaps due to slower protein turnover. Elevation in CCS level is one of the most dramatic alterations in Cu binding proteins accompanying Cu deficiency and may be useful to assess Cu status.

Copper, Zinc-Superoxide Dismutase Protein but not mRNA Is Lower in Copper-Deficient Mice and Mice Lacking the Copper Chaperone for Superoxide Dismutase

Cu, Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. Previous work with mice lacking the Cu chaperone for SOD1 (CCS) indicated a drastic loss of SOD1 activity but not protein, suggesting an accumulation of apo-SOD1. We produced dietary Cu deficiency in mice to clarify this issue. Compared with Cu-deficient rats, reduction in liver SOD1 activity and protein was much less than Cu-deficient mouse dams and offspring. However, after perinatal Cu deficiency, 4-week-old mouse pups had lower levels of SOD1 activity and protein in liver and heart, but not brain, compared with Cu-adequate controls. Reduction in brain Cu was greater than liver. In CCS –t– mice, there was severe reduction in liver, heart, and brain SOD1 activity and protein. In fact, the reduction in activity was similar to the loss of protein. Neither Cu-deficient mouse liver nor CCS -/- mouse liver had altered SOD1 mRNA levels compared with control values. These results in mice are comparable with rats and suggest a posttranscriptional mechanism for reduction of SOD1 protein when Cu is limiting in SOD1.

On the role of γ-carboxyglutamic acid in calcium and phospholipid binding

Summary The vitamin K-dependent amino acid, γ-carboxyglutamic acid, is essential for calcium binding by prothrombin and blood clotting factor X. The studies reported here demonstrate that while γ-carboxyglutamic acid-containing peptides will bind calcium, a secondary/tertiary protein structure is also necessary to form the tight calcium binding sites which are required for binding these proteins to phospholipid surfaces.

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattusnorvegicus), albino mice (Mus musculus), Cp-/- mice, and adult humans (Homo sapiens) to investigate the copper-iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp-/- mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp-/- mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60-90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.

Phorbol ester stimulates hexose uptake by brain microvessel endothelial cells

Glucose uptake into cultured endothelial cells (EC) derived from brain microvessels was determined in the absence and presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), EGTA, the calcium ionophore A23187, and insulin. EC were obtained from dog and human (autopsy) brain microvessels and maintained in culture for up to four passages. Monolayers of EC were treated with TPA and other compounds immediately prior to harvesting for hexose uptake measurements using 3-O-[3H]methyl-D-glucose, 2-[3H]deoxy-D-glucose, or D-[3H]glucose. Typically, treatment with TPA (0.1-100 ng/ml) resulted in hexose uptake levels 2 to 3 times those of controls, although occasionally levels 5 to 10 times those of controls were observed. Similar stimulation was observed with all radiolabeled hexoses. Stimulation by TPA was greatest in primary or first passage cells and was greatly diminished in older cells. Neither chelation of extracellular calcium with EGTA nor the presence of both EGTA and A23187 in the culture medium prevented the stimulatory effect of TPA. Insulin (1200 ng/ml) failed to stimulate hexose uptake. Treatment with 100 ng/ml TPA did not alter the appearance of actin filaments in canine EC as visualized with rhodamine phalloidin. These results, in combination with other recent studies, suggest that blood-brain glucose transport may be regulated by phorbol ester-activated protein kinase C.

Anemic Copper-Deficient Rats, but Not Mice, Display Low Hepcidin Expression and High Ferroportin Levels

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.

Differential impact of copper deficiency in rats on blood cuproproteins

Sensitive blood biochemical markers of dietary copper status are not yet known. Rat models were used to investigate the response of severe copper deficiency in dams and pups by comparing abundance of several cuproproteins in erythrocytes, white blood cells, and platelets. The hypothesis tested was that copper deficiency would result in changes in abundance of cuproproteins in blood cells. Copper-deficient (CuD) Holtzman dams and pups had signs consistent with severe copper deficiency compared with copper-adequate controls including lower liver copper and hemoglobin levels and near total loss of plasma ceruloplasmin diamine oxidase activity. Copper-deficient erythrocytes had lower copper, zinc superoxide dismutase (SOD1) but higher copper metallochaperone for SOD1 (CCS) compared with copper-adequate, resulting in higher CCS/SOD1 levels. This ratio was more sensitive in CuD erythrocytes than CuD white cells and especially in CuD platelets. However, both white blood cells and platelets from CuD dams and pups had nearly nondetectable levels of cytochrome c oxidase subunit IV. Because isolation of relatively pure populations of erythrocytes and platelets is feasible, and reagents for immunoblot methods are available, determination of CCS/SOD1 and cytochrome c oxidase subunit IV protein levels may be useful to assess copper status of humans.

Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.