Augmented metalloproteinase activity and acute lung injury in copper-deficient rats.

Dietary copper is required for normal function of >30 mammalian enzyme systems. Copper deficiency causes a number of cardiovascular defects as well as impaired immune cell function. Little is known regarding the effects of copper deficiency on acute inflammatory responses, but this topic is relevant because many members of the Western population receive less than the recommended dietary allowance of copper. In the current studies, we investigated the effects of dietary copper deficiency on acute lung injury induced by intrapulmonary deposition of IgG immune complexes. Weanling male Long-Evans rats were fed diets either adequate (5.6 microg/g) or deficient (0.3 microg/g) in copper. IgG immune complex lung injury was greatly increased in copper-deficient rats as determined by lung vascular leakage of albumin and histopathology. However, no change was observed in either the lung content of tumor necrosis factor-alpha or lung neutrophil accumulation. Lungs from copper-deficient rats had much higher levels of matrix metalloproteinase (MMP)-2 and MMP-9 than did copper-adequate control animals. This increased activity was not attributable to alveolar macrophages or neutrophils. These data suggest that the augmented lung injury caused by copper deficiency is due to increased pulmonary MMP-2 and MMP-9 activity and not a generalized amplification of the inflammatory response.

Blood flow shear rates in arterioles of spontaneously hypertensive rats at early and established stages of hypertension.

Alterations of blood rheological properties can affect blood flow shear rates and therefore alter changes in the interactions between blood and vascular wall components during the development of hypertension. This study was done to evaluate alterations of blood flow shear rates in resistance vessels during the development of genetic hypertension in rats. In the current study, measurements were carried out on spontaneously hypertensive rats (SHR) during an early (3 weeks of age) and an established stage (12 weeks of age) of hypertension development. Age matched normotensive Wistar Kyoto (WKY) rats were used as controls. Intravital television microscopy was used to quantitate blood flow shear rates in first-(1A), second-(2A) and third-order (3A) arterioles of the cremaster muscle. In the young SHRs mean arterial blood pressure was not different from age matched WKY rats, but there was a significant increase of shear rate values in all observed (1A, 2A, 3A) arterioles of SHRs. However, shear rate values were significantly less in arterioles (1A, 2A, 3A) of SHRs with an established hypertension compared to the 3-week-old SHR group. We conclude that shear rates are elevated in resistance vessels prior to an increase in mean arterial pressure during the development of genetic hypertension. These results suggest that a change in blood rheology may cause a change in peripheral vascular resistance and thus contribute to the pathogenesis of hypertension.

Leukocyte-endothelial adhesion is impaired in the cremaster muscle microcirculation of the copper-deficient rat.

Dietary copper deficiency impairs the function of both the vascular endothelium and circulating leukocytes. In the current study, leukocyte-endothelium adhesion was observed in the in vivo cremaster muscle microcirculation of copper-adequate and copper-deficient rats. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (5.6 microg/g) or deficient (0.3 microg/g) in copper. Adhesion was stimulated with the inflammatory mediators tumor necrosis factor-alpha and bradykinin, and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. Intravascular adhesion of leukocytes to the vascular endothelium was significantly attenuated in the copper-deficient group in response to all three agonists. These results occurred without any difference in intravascular wall shear rate between the dietary groups. Based on previous work, we propose that the attenuated response is caused by either decreased expression of adhesion molecules on leukocytes and endothelial cells or by inhibition of the endothelial cell calcium signaling associated with copper deficiency.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

The effect of severe burn injury on proinflammatory cytokines and leukocyte behavior: its modulation with granulocyte colony-stimulating factor.

Severe injury causes immunosuppression. The main contributors are impaired leukocyte function and a cytokine dysbalance. GCSF increases PMN count, function and modulates the inflammatory response. However GCSF may overactivate leukocytes. The purpose of this study is to investigate whether GCSF is able to restore immune competence after severe injury. Lewis rats were divided into three groups: 30% TBSA burn + vehicle; 30% TBSA burn + GCSF (150 microg rhGCSF); Control. Blood samples were taken for total white cell count, PMNs, TNFalpha and IFNgamma. Leukocyte rolling and sticking were measured in the cremaster muscle microcirculation. Leukocyte diapedesis was investigated by lavage of the abdominal cavity and the lungs. Total white cell and PMN counts in the burn + GCSF group were significantly higher (P<0.001) than in burn+vehicle animals. Leukocyte adherence and diapedesis were not elevated in the burn + GCSF group as compared to the burn + vehicle group. TNFalpha (P<0.05) and IFNgamma (P<0.001) levels were significantly increased in the burn + vehicle animals compared to the burn + GCSF animals. GCSF modifies the immune system, as shown by an increase in white cell and PMN counts and by balancing the overall immune response from proinflammatory to normal, as shown by decreased TNFalpha and IFNgamma levels. GCSF does not overactivate PMNs.

Microcirculatory changes following different temperature washouts in a free flap model.

In spite of the extensive experimental work on vascular washout in free flap surgery, an optimal temperature for the washout solution has not been established. This study was designed to determine the effect of the washout solution temperature on the degree to which the microcirculation is cleared of blood. The cremaster muscle flap in the rat was used, in which the microcirculation can be directly viewed and the presence of blood and perfusion parameters within various vessels can be measured during and after washout. Washout was started with a single, high-pressure infusion and continued at 130 mmHg for 15 minutes. The temperature of the washout solution was either 2-3, 20-22, or 35 degrees C. In all three groups, washout cleared the microcirculation almost completely within the first minute. However, we observed that a cold or room temperature washout cleared the microcirculation more completely than a warm washout did. The temperature of the washout solution did not effect post washout capillary perfusion and/or arterial diameters.

Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity.

To study cellular and molecular events of cardiac protection by metallothionein (MT) from oxidative injury, a primary neonatal cardiomyocyte culture was established from a specific cardiac MT-overexpressing transgenic mouse model. Ventricular cardiomyocytes were isolated from 1- to 3-day-old neonatal mice and cultured in an Eagle's minimum essential medium supplemented with 20% fetal bovine serum under an atmosphere of 5% CO2-95% air at 37 degreesC. Forty-eight hours after plating was completed, the purity of such cultures was 95% myocytes, assessed by an immunocytochemical assay. Over 80% of the cardiomyocytes beat spontaneously on the first day of culture and synchronously in a confluent monolayer after the sixth day of culture. Cellular MT concentrations in the transgenic cardiomyocytes before culturing and on the sixth day postculturing were about seven- and twofold higher than nontransgenic controls, respectively. However, there were no significant differences in cell morphology, glutathione content, and antioxidant enzymatic activities between these two types of cardiomyocytes. When these cells were challenged by H2O2, the transgenic cardiomyocytes displayed a significant resistance to the toxic effect of this oxidant, as measured by cell viability, lactate dehydrogenase leakage, and morphological alterations. In addition, the transgenic cells were highly protected from H2O2-induced lipid peroxidation. These observations demonstrate that MT protects the cultured cardiomyocytes from H2O2 toxicity by preventing its interaction with macromolecules such as lipids, and this cultured primary neonatal mouse cardiomyocyte system provides a valuable tool to directly study cellular and molecular events of MT in cardiac protection against oxidative injury.

Relationship between dietary copper concentration and acetylcholine-induced vasodilation in the microcirculation of rats.

Dietary copper deficiency has been shown to significantly reduce acetylcholine (Ach)-induced vascular smooth muscle relaxation. The current study was designed to examine the relative relationship between dietary copper and the vasodilator response to Ach in the microcirculation of the rat. Male weanling rats were fed a purified basal diet supplemented with 6.0, 3.0, 1.5 or 0.0 microg Cu/g diet for 4 weeks to provide an adequate, two marginal, and deficient intakes of dietary copper. Arteriole dilation in response to increasing concentrations of acetylcholine (10(-7) to 10(-4) M) was measured in the in vivo cremaster muscle microcirculation for each dietary group. Liver copper and both aortic and erythrocyte Cu,Zn-SOD activity were used as indices of systemic copper status. Dilation to the increasing concentrations of Ach was only different in the 0 microg Cu supplemented group compared to the copper-adequate control values. However, the combined results showed an exponential increase in 10(-5) M Ach-induced vasodilation as liver copper concentration increases from 0 microg Cu/g dry wt. This relationship suggests that dilation is attenuated at liver Cu concentrations below 5 microg/g dry wt. The results indicate that Ach-induced vasodilation is copper-dependent but that the pathway is not very sensitive to short-term marginal restriction of copper intake.

Cardiovascular effects of dietary copper deficiency.

Dietary copper deficiency may impair cardiovascular health by contributing to high blood pressure, enhancement of inflammation, anemia, reduced blood clotting and arteriosclerosis. The purpose of this review is to compile information on the numerous changes of the heart, blood and blood vessels that may contribute to these cardiovascular defects. These alterations include weakened structural integrity of the heart and blood vessels, impairment of the use of energy by the heart, reduced ability of the heart to contract, altered ability of blood vessels to control their diameter and to grow, and altered structure and function of circulating blood cells. The fundamental causes of these changes rest largely on reduced effectiveness of enzymes that depend on copper for their activity.

Platelet thrombus formation in microvessels of young spontaneously hypertensive rats.

We have previously shown an increase in platelet-to-endothelial cell adhesion in microvessels of spontaneously hypertensive rats (SHR) during the established stage of hypertension (12 weeks). The objective of the current study was to determine if the platelet-to-endothelial cell interaction would be altered in the early developmental phase of hypertension. Male weanling (3 weeks old) SHRs (n=6) and age matched normotensive Wistar-Kyoto (WKY) rats (n=6) were used to study platelet thrombus formation. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Plasma concentrations of von Willebrand factor (vWF), fibrinogen and fibronectin (FN) were measured in rats during both early (3 week) and established stages of hypertension development. Thrombus initiation time in both arterioles (847+/-85 sec) and venules (222+/-40 sec) of young SHRs was significantly shorter (p<0.05) than in arterioles (1270+/-88 sec) and venules (630+/-72 sec) of age matched WKY rats respectively. After thrombus appearance, however, overall time for vessel occlusion in arterioles (2590+/-90 sec) and venules (935+/-131 sec) of SHRs was not different compared to that in arterioles (2650+/-191 sec) and venules (1240+/-93 sec) of age matched WKY rats. The plasma concentration of FN was increased (p<0.05) in both the young (0.9+/-0.1 mg/ml) and mature (1.1+/-0.2 mg/ml) hypertensive rats (n=5) compared to that in young (0.6+/-0.03 mg/ml) and mature (0.5+/-0.1 mg/ml) WKY rats (n=5), while fibrinogen content (3.6 +/-0.3 mg/ml) was elevated (p<0.05) only in mature SHRs (n=5) compared to that (2.7+/-0.02 mg/ml) in age matched WKY rats (n=5). The plasma concentration of vWF was similar to that of controls in either age group of hypertensive animals. These results suggest that changes in platelet-to-endothelial cell interactions occur in the early phase of genetic hypertension development in rats, and appears to result from alteration of plasma concentration of adhesion proteins.

Leukocyte behavior in a free-flap model following chemotherapy and application of granulocyte colony-stimulating factor (GCSF).

Free-flap reconstruction following tumor resection and chemotherapy is used increasingly in sarcoma patients. Granulocyte colony-stimulating factor (GCSF) is used to stimulate polymorphnuclear leukocytes (PMNs). In this study we used a free-flap model to investigate PMN behavior after chemotherapy and GCSF stimulation. Eighteen Lewis rats were divided into three groups: (1) chemotherapy + vehicle; (2) chemotherapy + GCSF; and (3) control. Group 1 received vinblastine; group 2 received vinblastine plus 300 microg rhGCSF. Blood was drawn for total white cell and PMN counts for 9 days. Free-flap surgery was simulated by isolating the cremaster muscle on its pedicle and anastomosing the artery. Leukocyte-endothelium interaction was assessed by observation of leukocytes rolling and sticking. Leukocyte sequestration was measured by counting leukocytes in the lungs and the abdomen. We found that leukocyte rolling and sticking were significantly increased, while sequestration was decreased in the chemotherapy + GCSF group. We conclude that leukocytes-endothelium interaction after chemotherapy and GCSF administration is increased in the microcirculation. This augments the risk of microvascular compromise and subsequent flap failure, as capillary flow may be altered.

Augmentation of skeletal muscle flap survival using platelet derived growth factor.

Distal muscle flap ischemia and necrosis is a recognized complication of acute elevation of large skeletal muscle flaps. The aim of this study was to investigate whether the angiogenic properties of platelet derived growth factor (PDGF) could be used to augment skeletal muscle flap survival through the induction of new blood vessel formation before flap elevation. We compared this form of flap augmentation with that achieved by subjecting the muscle to a bipedicled vascular delay procedure. The animal model used was the latissimus dorsi muscle of the male homozygous (hr/hr) hairless mouse. Four groups of animals were investigated in this study (n = 10 per group). Group 1 was the control group in which the entire muscle was elevated as a thoracodorsally based island flap. In group 2, the muscle was subjected to a bipedicled vascular delay procedure. In group 3, the muscle was treated with 500 microg of recombinant human platelet derived growth factor BB. In group 4, the muscle was treated with placebo. Ten days later the entire latissimus dorsi muscle was elevated as a thoracodorsally based island flap in groups 2, 3, and 4. Percentage muscle flap survival was quantitated in all groups 5 days after elevation of the entire muscle. Angiogenesis was then quantitated by analyzing capillary to muscle fiber ratios after alkaline phosphatase staining of representative latissimus dorsi muscle samples from the proximal, middle, and distal flap segments. Percentage muscle flap survival was significantly better in PDGF treated muscles when compared with the vascularly delayed muscles (p < 0.001). Histologic analysis of latissimus dorsi muscle flaps demonstrated a significantly greater number of capillaries in the middle (p < 0.001) and distal (p < 0.001) flap segments of PDGF-treated flaps when compared with the vascularly delayed flaps. Treatment of skeletal muscle with PDGF before flap creation resulted in survival of the entire muscle flap. Our results suggest that this survival may be secondary to PDGF-induced angiogenesis.

Increased erythrocyte aggregation in spontaneously hypertensive rats.

Alterations of red blood cell (RBC) aggregation and plasma viscosity are major contributors to the changes in blood rheologic properties that cause an increase in peripheral vascular resistance during the development of hypertension. Although basic research and clinical study have provided considerable understanding of the pathophysiology of hypertension, the objective of this study was to determine whether an increase in RBC aggregability and plasma viscosity precede or accompany the development of high arterial blood pressure. To address this question, RBC aggregation and plasma viscosity were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 3 and 12 weeks of age. The plasma concentrations of fibrinogen and fibronectin (FN) were also analyzed in both age groups. RBC aggregability and plasma viscosity were increased in both young and mature SHR compared to age-matched normotensive WKY rats. Mean arterial blood pressure and diastolic pressures were increased in mature hypertensive rats, whereas in young SHR only diastolic pressure was elevated significantly. The concentration of fibrinogen was higher only in the mature hypertensive rats, whereas plasma FN content was greater in both 3- and 12-week-old SHR compared to age-matched WKY. These results show the existence of increased RBC aggregability and plasma hyperviscosity not only during the established phase of hypertension, but also during the early stage of hypertension development, when mean arterial blood pressure is not yet significantly elevated in the genetically hypertensive rat model. These changes may be related to significant increase in the plasma protein FN, which occurs at the same time as the RBC aggregability and plasma viscosity changes. These results may increase attention to changes in the rheologic properties and to the mechanisms involved in these processes in the early stages of hypertension development.

Do leukocytes contribute to impaired microvascular tissue perfusion after arterial repair?

Impaired capillary perfusion may result in flap failure. Platelet emboli, polymorphonuclear leukocytes (PMNs), and/or vasospasm have been identified as possible causes. This study investigates the role of PMNs in causing impaired capillary perfusion in a free flap model. PMN concentrations were depleted using antineutrophil serum. The cremaster muscles of 20 Sprague-Dawley rats were isolated on a single neurovascular pedicle and after a simulated technically poor arterial anastomosis upstream and reperfusion, capillary perfusion was measured each hour for 6 hours. Even though the number of PMNs was significantly reduced in the animals treated with antineutrophil serum, capillary perfusion was not changed compared with controls. These results demonstrate that depleting circulating PMNs does not protect capillary perfusion in our model. These findings suggest that reduced capillary perfusion downstream from an anastomotic repair is not mediated by the presence of PMNs in the microcirculation.

In vivo platelet thrombus formation in microvessels of spontaneously hypertensive rats.

Sustained high blood pressure causes functional changes in both vascular endothelial cells and platelets. Therefore, we hypothesized that in vivo platelet thrombus formation would be increased in the cremaster muscle microvessels of rats during genetic hypertension. Experiments were carried out on spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) at 12 weeks of age. Fluorescein isothiocyanate tagged to bovine serum albumin (FITC-BSA) was injected intraarterially and 450 to 490 nm light was used to activate the FITC-BSA and induce a thrombus within the vasculature. In vivo television microscopy was used to quantitate thrombus formation and microvascular diameter changes. The time of platelet thrombus initiation and subsequent time of thrombus growth were studied at wall shear rates of approximately 2000 sec(-1) and 270 sec(-1) in third-order arterioles and venules, respectively. In SHR, times for platelet thrombus initiation and vessel occlusion were significantly less in both arterioles and venules, whereas time for platelet thrombus growth following initiation was significantly prolonged. Greater shear rates in arterioles compared to venules decreased platelet adhesion and subsequently decreased the rate of thrombus formation in both WKY and SHR groups. However, the ratio of WKY to SHR platelet thrombus growth (platelet aggregation) time remained similar (0.83 +/- 0.06 in arterioles and 0.79 +/- 0.06 in venules). These results indicate that there is increased thrombus formation during hypertension and that the platelet adhesion processes may be of greater importance than platelet aggregation in producing this increase.

Von Willebrand factor restores impaired platelet thrombogenesis in copper-deficient rats.

Dietary copper restriction reduces microvascular thrombogenesis. We have now examined the roles of shear forces and von Willebrand factor (vWF) in in vivo thrombus formation in the cremaster microcirculation of copper-deficient rats. Male weanling Sprague-Dawley rats were fed purified diets that were either copper-adequate (6.3 mg Cu/kg) or copper-deficient (0.3 mg Cu/kg) for 4 wk. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Thrombus initiation time was significantly prolonged in copper-deficient rats; after thrombus appearance, however, vessel occlusion was significantly accelerated. The greater shear rates of arterioles compared with venules significantly increased the thrombus initiation time in both groups. However, vessel occlusion time and thrombus growth time were independent of shear rate. Intravascular vWF (0.2 u/100 g body wt) decreased thrombus initiation time in the CuD group without affecting thrombus growth time. The data suggest that decreased thrombogenesis in copper-deficient rats is not a result of altered rheological factors or arteriolar-venular differences, but appears to result from decreased platelet-to-endothelial cell adhesion.

Antithrombotic and vasorelaxant properties of a novel synthetic RGD peptide containing nitric oxide.

We have developed a novel synthetic peptide containing both the antiadhesive Arg-Gly-Asp (RGD) amino acid sequence and a nitric oxide (NO) moiety well known for its vasorelaxant properties. The main objective of this study was to determine whether this hybrid molecule is concurrently effective with regard to antithrombotic and vasorelaxation actions. Studies of in vitro platelet adhesion and of in vivo platelet thrombus formation in the rat demonstrated that the RGD-NO peptide increased the antithrombotic characteristics of the RGD peptide alone. The RGD-NO peptide also caused relaxation of rat aortic rings, while the RGD peptide did not induce relaxation. These characteristics of Ac-RGDC-SNO suggest that this or similar compounds may have potential as effective antithrombotic agents in coronary and peripheral artery disorders.

Effect of low dose aspirin on thrombus formation at arterial and venous microanastomoses and on the tissue microcirculation.

In free flap/replantation surgery, failure is usually associated with thrombotic occlusion of a microvascular anastomosis (risk zone I) or, on occasion, flow impairment in the microcirculation of the transferred or replanted tissue (risk zone II). The objective of this study is to describe the effect of low dose aspirin on blood flow at both risk zones in microvascular surgery. Risk zone I: In rat femoral arteries and veins, thrombus formation was measured at the anastomoses using transillumination and videomicroscopy. Forty male Wistar rats were assigned in equal numbers to four groups: either arterial or venous injury with either aspirin (5 mg/kg systemically) or saline treatment. We found that aspirin significantly reduces thrombus formation at the venous anastomosis (p = 0.001). Risk zone II: In the isolated rat cremaster muscle downstream from an arterial anastomosis, we measured capillary perfusion, arteriolar diameters, and the appearance of platelet emboli for 6 hours in the muscle microcirculation. Sixteen male Wistar rats in two equal groups received either aspirin (5 mg/kg systemically) or saline. We found that in aspirin-treated animals, capillary perfusion is significantly (p = 0.002) improved, whereas arteriolar diameters and emboli only slightly increased. In conclusion, low dose aspirin inhibits anastomotic venous thrombosis and improves microcirculatory perfusion in our rat model. These studies provide quantitative data confirming and clarifying the beneficial effects of low dose aspirin in microvascular surgery.

Arteriolar dilation to endotoxin is increased in copper-deficient rats.

We have previously reported that there is an altered response to mast cell-mediated inflammation in copper-deficient rats. In the current study we determined the microvascular reactivity to inflammatory stimuli with lipopolysacccharide (LPS) during dietary copper restriction. Male Sprague-Dawley rats were fed purified diets which were either copper-adequate (CuA, 6 micrograms Cu/g) or copper-deficient (CuD, 0.4 micrograms Cu/g) for 4 weeks. Rats were anesthetized and the cremaster muscle was prepared for in vivo television microscopy. Arteriolar diameters were measured and then 2.5 mg/kg LPS was injected i.p. In separate groups, animals were pretreated with the NO-synthase inhibitor L-NAME (2 x 10(-4) M), the cyclooxygenase inhibitor ibuprofen (9.6 x 10(-5) M) or the histamine receptor antagonist diphenhydramine (DPH, 10(-6) M). LPS caused arteriolar dilation in both dietary groups with the response being significantly greater in the CuD group. Ibuprofen and DPH but not L-NAME, each significantly reduced but did not block the dilation in the CuD group. Ibuprofen and DPH together blocked the dilation. These results suggest that dietary copper deficiency increases arteriolar dilation to LPS. The mechanism appears to involve a greater response to arachidonic acid metabolites and histamine but not NO.

Dietary copper in the physiology of the microcirculation.

Dietary copper has long been known to be essential for cardiovascular homeostasis. However, the role of copper and cuproenzymes in the normal control of vascular physiology is not well understood. Most studies in the cardiovascular system have focused on copper deficiency-induced defects in the heart or large vessels. Recently, attention has also focused on the effects of copper deficiency in the microcirculation or the small blood vessels that control blood flow, nutrient and waste exchange, and peripheral vascular resistance. Studies in the microcirculation demonstrate that copper is important in mechanisms of macromolecular leakage, platelet-endothelial interactions and vascular smooth muscle reactivity. There is a significantly greater leakage of proteins from postcapillary venules in copper-deficient rats in response to mast cell-released histamine. This response appears to be the result of increased numbers of mast cells and thereby increased available histamine. Copper deficiency also causes an inhibition of in vivo thrombogenesis, which appears to be related to an inhibition of platelet adhesion. Subsequent studies have demonstrated that this is probably caused by a diminished concentration of the adhesion molecule von Willebrand factor. Nitric oxide (NO)-mediated arteriole vasodilation is also compromised in copper-deficient rats. This functional deficit to NO can be reversed by the addition of Cu, Zn-superoxide dismutase (SOD), suggesting that degradation of NO by superoxide anion occurs during copper deprivation. These observations demonstrate that dietary copper is necessary for several microvascular control mechanisms affecting inflammation, microhemostasis and regulation of peripheral blood flow.