Sialyl Lewisx-containing oligosaccharide attenuates myocardial reperfusion injury in cats.

Neutrophil (PMN) adhesion to the vascular endothelium is an important mechanism of myocardial reperfusion injury. The adhesion process is initially mediated by selectins (e.g., P- and L-selectin), and monoclonal antibodies directed against these adhesion molecules exert cardioprotective activity in ischemia/reperfusion models. The counterreceptors for these selectins are thought to be carbohydrate-containing moieties. In this connection, we studied the effect of a soluble sialyl Lewisx-containing oligosaccharide (SLex-OS) on PMN-endothelial interactions in a feline model of myocardial ischemia/reperfusion (MI/R). SLex-OS (10 mg/kg), administered 10 min before R, significantly reduced myocardial necrosis compared with its vehicle 270 min after reperfusion (6 +/- 1% vs. 35 +/- 4% of area at risk, P < 0.01). The cardioprotection was confirmed by significantly lower plasma creatine kinase activities in SLex-OS vs. vehicle-treated cats (P < 0.01). Cardiac contractility (dP/dt max) of cats receiving SLex-OS was significantly preserved after 270 min of R (97 +/- 2% vs. 78 +/- 5% of initial, P < 0.01). Furthermore, endothelium-dependent relaxation to acetylcholine in coronary artery rings isolated from MI/R cats treated with SLex-OS was significantly preserved (73 +/- 7% vs. 22 +/- 6% vasorelaxation, P < 0.01). In vitro PMN adherence to coronary vascular endothelium after 270 min of R was significantly attenuated in the SLex-OS-treated group compared with the vehicle group (14 +/- 5 vs. 91 +/- 12 PMN/mm2, P < 0.01). Our results indicate that a SLex-OS is cardioprotective and preserves coronary endothelial function after MI/R, indicating an important role of sialyl Lewisx in PMN accumulation, endothelial dysfunction, and myocardial injury in myocardial ischemia/reperfusion.     

Intercellular adhesion molecule 1 is important for the development of severe experimental malaria but is not required for leukocyte adhesion in the brain.

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit a systemic inflammatory response. Most investigators hypothesize that leukocytes bind to endothelial cells via intercellular adhesion molecule 1 (ICAM-1), which causes endothelial damage, increased microvascular permeability, and, ultimately, death. ICAM-1-deficient mice on an ECM-susceptible C57BL/6 background were significantly (p = .04) protected from P. berghei mortality compared with ICAM-1 intact controls. ICAM-1 expression assessed by the dual radiolabeled monoclonal antibody technique was increased in the brain and lung in C57BL/6 mice on day 6 of P. berghei infection compared with uninfected controls (5.3-fold, p = .0003 for brain and 1.8-fold, p = .04 for lung). The increase in ICAM-1 expression coincided with significant (p < .05) increases in microvascular permeability in the brain and lung. In contrast to the hypothesized role for ICAM-1, in vivo analysis by intravital microscopy of leukocyte rolling and adhesion in brain microvasculature of mice revealed markedly increased levels of leukocyte rolling and adhesion in ICAM-1-deficient mice on day 6 of P. berghei infection compared with uninfected controls. In addition, ICAM-1 expression and microvascular permeability were increased in infected ECM-resistant BALB/c mice compared with uninfected BALB/c controls. These results collectively indicate that although ICAM-1 contributes to the mortality of experimental malaria, it is not sufficient for the development of severe experimental malaria. In addition, ICAM-1 expressed on the endothelium or on leukocytes is not required for leukocyte rolling or adhesion to the brain microvasculature of mice during P. berghei malaria. Leukocyte rolling and adhesion in the brain vasculature during P. berghei malaria use different ligands than observed during inflammation in other vascular beds.     

Intercellular adhesion molecule-1 is upregulated on peripheral blood T lymphocyte subsets in dual asthmatic responders.

To examine the role of adhesion molecules in T cell recruitment and activation during allergen-induced late asthmatic response (LAR), we evaluated the expression of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) on peripheral blood T lymphocyte subsets from atopic asthmatic patients and their changes following allergen inhalation challenge. 12 atopic asthmatic patients were studied. Six patients showed only a single early response after allergen challenge, and six developed a dual response. At baseline, dual responders (DR) had a significantly higher expression of ICAM-1 on CD4+ and CD8+ T lymphocytes as compared with both single early responders (P < 0.005 and P < 0.02, respectively) and controls (P < 0.001, both comparisons). Allergen challenge was followed by a decrease of CD8+ ICAM-1+ T lymphocytes in all DR (P < 0.05) and of CD4+ ICAM-1+ T lymphocytes in four out of six DR, at the time of the LAR. At the same time, a significant rise in serum levels of the soluble form of ICAM-1 was observed in DR. These results suggest that peripheral blood immunoregulatory T lymphocytes are in a higher state of activation in DR as compared with early responders. The upregulation of ICAM-1 on these cells may be important in enhancing airway inflammation in patients with LAR.     

Chimeric mice carrying 'regional' targeted deletion of the angiotensin type 1A receptor gene. Evidence against the role for local angiotensin in the in vivo feedback regulation of renin synthesis in juxtaglomerular cells.

We have developed chimeric mice carrying 'regional' null mutation of the angiotensin type 1A (AT1A) receptor, the AT1 receptor subtype exclusively present in mouse juxtaglomerular (JG) cells. The chimeric mouse (Agtr1a -/- <--> +/+) is made up of wild-type (Agtr1a +/+) cells or cells homozygous for Agtr1a deletion (Agtr1a -/-). In the latter, the AT1A coding exon was replaced with a reporter gene, lacZ. In Agtr1a -/- <--> +/+ mice, these two clones of cells are found to be clustered and display patchy distributions in the kidney and heart. Tracking of lacZ activities in hetero- (Agtr1a +/-) and homozygous (Agtr1a -/-) deletion mutant offspring from Agtr1a -/- <--> +/+ mice revealed that the promoter activity of Agtr1a is localized in JG cells, afferent arteriolar walls, glomerular mesangial region and endothelial cells, and apical and basolateral proximal tubule membranes. The JG apparatuses of Agtr1a -/- mice are markedly enlarged with intense expression of renin mRNA and protein. In Agtr1a -/- <--> +/+ mice, these changes were proportional to the degree of chimerism. Within a given Agtr1a -/- <--> +/+ mouse, however, the degree of JG hypertrophy/hyperplasia and the expression of renin mRNA and protein were identical between Agtr1a +/+ and Agtr1a -/- cells. Thus, in the in vivo condition tested, the local interaction between angiotensin and the AT1 receptor on the JG cells has little functional contribution to the feedback regulation of JG renin synthesis.     

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.     

Reduced microvascular thrombosis and improved outcome in acute murine stroke by inhibiting GP IIb/IIIa receptor-mediated platelet aggregation.

Treatment options in acute stroke are limited by a dearth of safe and effective regimens for recanalization of an occluded cerebrovascular tributary, as well as by the fact that patients present only after the occlusive event is established. We hypothesized that even if the site of major arterial occlusion is recanalized after stroke, microvascular thrombosis continues to occur at distal sites, reducing postischemic flow and contributing to ongoing neuronal death. To test this hypothesis, and to show that microvascular thrombosis occurs as an ongoing, dynamic process after the onset of stroke, we tested the effects of a potent antiplatelet agent given both before and after the onset of middle cerebral arterial (MCA) occlusion in a murine model of stroke. After 45 min of MCA occlusion and 23 h of reperfusion, fibrin accumulates in the ipsilateral cerebral hemisphere, based upon immunoblotting, and localizes to microvascular lumena, based upon immunostaining. In concordance with these data, there is a nearly threefold increase in the ipsilateral accumulation of 111In-labeled platelets in mice subjected to stroke compared with mice not subjected to stroke. When a novel inhibitor of the glycoprotein IIb/IIIa receptor (SDZ GPI 562) was administered immediately before MCA occlusion, platelet accumulation was reduced 48%, and fibrin accumulation was reduced by 47% by immunoblot densitometry. GPI 562 exhibited a dose-dependent reduction of cerebral infarct volumes measured by triphenyltetrazolium chloride staining, as well as improvement in postischemic cerebral blood flow, measured by laser doppler. GPI 562 caused a dose-dependent increase in tail vein bleeding time, but intracerebral hemorrhage (ICH) was not significantly increased at therapeutic doses; however, there was an increase in ICH at the highest doses tested. When given immediately after withdrawal of the MCA occluding suture, GPI 562 was shown to reduce cerebral infarct volumes by 70%. These data support the hypothesis that in ischemic regions of brain, microvascular thrombi continue to accumulate even after recanalization of the MCA, contributing to postischemic hypoperfusion and ongoing neuronal damage.     

Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats.

Peroxynitrite (ONOO-) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO- on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO- on PMN-mediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin- or H2O2-stimulated rat mesenteric artery (P < 0.01 vs. thrombin or H2O2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ml) or N(G)-nitro-L-arginine-methyl ester (50 microM) significantly increased venular leukocyte rolling and adherence, which were also significantly (P < 0.01) attenuated by ONOO (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10(8) cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO- reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO- both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO- on leukocyte-endothelial cell interactions.     

In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.     

Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury.

Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.     

Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.

To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.     

Interleukin-1 receptor antagonist ameliorates experimental anti-glomerular basement membrane antibody-associated glomerulonephritis.

The contribution of IL-1 to leukocyte infiltration in anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was examined by the administration of a specific IL-1 receptor antagonist (IL-1ra). Lewis rats received anti-GBM Ab or normal rabbit serum and were treated with either 0.9% saline or 6 mg IL-1ra over a 24-h time period. Plasma IL-1ra concentration was 2,659 +/- 51 ng/ml 4 h after anti-GBM Ab and IL-1ra administration. PMN and monocyte/macrophage infiltration declined 39% (9.8 +/- 1.9 to 6.0 +/- 1.5 PMN/glomerulus, P < 0.001) and 29% (4.9 +/- 0.8 to 3.5 +/- 0.8 ED-1 cells/glomerulus, P = 0.002) with IL-1ra treatment at 4 h, respectively. Similarly, the number of glomerular cells staining for lymphocyte function-associated molecule-1 beta (CD18) declined 39% from 16.7 +/- 1.9 to 10.7 +/- 1.6 cells/glomerulus at 4 h (P = 0.0001). This was associated with a decrease in glomerular intracellular adhesion molecule-1 expression. The mean glomerular intracellular adhesion molecule-1 score in anti-GBM Ab GN rats treated with IL-1ra was less than that of rats administered anti-GBM Ab and 0.9% saline at 4 (2.0 +/- 0.2 vs 2.5 +/- 0.2, P < 0.05) and 24 (2.5 +/- 0.1 vs 3.1 +/- 0.2, P = 0.0001) h. These immunopathologic changes correlated with a 50% reduction in proteinuria from 147 +/- 34 to 75 +/- 25 mg/d (P < 0.002). Treatment with IL-1ra did not affect the steady state mRNA expression of either IL-1 beta or TNF alpha. An increase in the IL-1ra dose to 30 mg given within the initial 4 h provided no additional benefit. The decline in PMN and monocyte/macrophage infiltration of the glomerulus at 4 h was similar to that found in the initial study. Furthermore, the protective benefit of IL-1ra was abrogated by doubling the dose of the anti-GBM Ab GN, despite administering high dose IL-1ra (30 mg). In these studies, detectable IL-1ra was found in the serum of untreated anti-GBM Ab GN controls. These data suggest a positive yet limited role for IL-1ra in the therapeutic intervention of anti-GBM Ab GN.     

Tumor necrosis factor-alpha blockade prevents neutrophil CD18 receptor upregulation and attenuates acute lung injury in porcine sepsis without inhibition of neutrophil oxygen radical generation.

Tumor necrosis factor (TNF alpha), both by direct action and by trafficking cells of the immune system, is implicated in cardiopulmonary derangements and PMN-mediated microvascular injury associated with gram-negative sepsis. We examined the effects of pretreatment with a monoclonal antibody to TNF alpha on PMN function, hemodynamic derangements, and alveolar capillary membrane damage in a septic porcine model. Anti-TNF alpha profoundly improved hemodynamic consequences in this model. Reduction in PMN CD11/18 receptor expression, lung myeloperoxidase activity, and attenuation of peripheral neutropenia (all P < 0.05) indicate that pretreatment significantly reduced lung sequestration of PMNs seen in septic controls. In contrast, PMN oxygen radical (O2-) generation was not significantly different from unprotected septic animals. Despite the presence of circulating PMNs primed for O2- burst, alveolar capillary membrane damage, assessed by bronchoalveolar lavage protein content and arterial PO2 was markedly attenuated in the treatment group (P < 0.05). We conclude that anti-TNF alpha suppresses systemic hemodynamic actions of TNF alpha. Further, it prevents upregulation of PMN adhesion receptors inhibiting PMN/endothelial cell interaction. This prevents formation of a "microenvironment," protected from circulating oxidant scavengers, into which sepsis-activated PMNs release their toxic products. Pretreatment with anti-TNF alpha monoclonal antibody thus affords global protection in porcine Gram-negative sepsis.     

Increased epinephrine and skeletal muscle responses to hypoglycemia in non-insulin-dependent diabetes mellitus.

We evaluated skeletal muscle counterregulation during hypoglycemia in nine subjects with non-insulin-dependent diabetes mellitus (NIDDM) (HbA1c 9.4 +/- 0.5%, nl < 6.2%) compared with six normal controls, matched for age (51 +/- 3 and 49 +/- 5 yr, respectively) and body mass index (27.3 +/- 1.2 and 27.0 +/- 2.1 kg/m2). After 60 min of euglycemia (plasma insulin approximately 140 microU/ml), plasma glucose was lowered to 62 +/- 2 mg/dl by 120 min. Hypoglycemia induced a 2.2-fold greater increase in plasma epinephrine in NIDDM (P < 0.001), while the plasma glucagon response was blunted (P < 0.01). Hepatic glucose output ([3H-3]glucose) suppressed similarly during euglycemia, but during hypoglycemia was greater in NIDDM (P < 0.005). Conversely, glucose uptake during euglycemia was 150% greater in controls (P < 0.01) and remained persistently higher than in NIDDM during hypoglycemia. In NIDDM, plasma FFA concentrations were approximately fivefold greater (P < 0.001), and plasma lactate levels were approximately 40% higher than in controls during hypoglycemia (P < 0.01); the rates of glycolysis from plasma glucose were similar in the two groups despite a 49% lower rate of glucose uptake in NIDDM (3.4 +/- 0.9 vs. 6.9 +/- 1.3 mg/kg per minute, P < 0.001). Muscle glycogen synthase activity fell by 42% with hypoglycemia (P < 0.01) in NIDDM but not in controls. In addition, glycogen phosphorylase was activated by 56% during hypoglycemia in NIDDM only (P < 0.01). Muscle glucose-6-phosphate concentrations rose during hypoglycemia by a twofold greater increment in NIDDM (P < 0.01). Thus, skeletal muscle participates in hypoglycemia counterregulation in NIDDM, directly by decreased removal of plasma glucose and, indirectly, by providing lactate for hepatic gluconeogenesis. Consequently, in addition to inherent insulin resistance in NIDDM, the enhanced plasma epinephrine response during hypoglycemia may partially offset impaired glucagon secretion and counteract the effects of hyperinsulinemia on liver, fat, and skeletal muscle.     

Mechanism of enhanced insulin sensitivity in athletes. Increased blood flow, muscle glucose transport protein (GLUT-4) concentration, and glycogen synthase activity.

We examined the mechanisms of enhanced insulin sensitivity in 9 male healthy athletes (age, 25 +/- 1 yr; maximal aerobic power [VO2max], 57.6 +/- 1.0 ml/kg per min) as compared with 10 sedentary control subjects (age, 28 +/- 2 yr; VO2max, 44.1 +/- 2.3 ml/kg per min). In the athletes, whole body glucose disposal (240-min insulin clamp) was 32% (P < 0.01) and nonoxidative glucose disposal (indirect calorimetry) was 62% higher (P < 0.01) than in the controls. Muscle glycogen content increased by 39% in the athletes (P < 0.05) but did not change in the controls during insulin clamp. VO2max correlated with whole body (r = 0.60, P < 0.01) and nonoxidative glucose disposal (r = 0.64, P < 0.001). In the athletes forearm blood flow was 64% greater (P < 0.05) than in the controls, whereas their muscle capillary density was normal. Basal blood flow was related to VO2max (r = 0.63, P < 0.05) and glucose disposal during insulin infusion (r = 0.65, P < 0.05). The forearm glucose uptake in the athletes was increased by 3.3-fold (P < 0.01) in the basal state and by 73% (P < 0.05) during insulin infusion. Muscle glucose transport protein (GLUT-4) concentration was 93% greater in the athletes than controls (P < 0.01) and it was related to VO2max (r = 0.61, P < 0.01) and to whole body glucose disposal (r = 0.60, P < 0.01). Muscle glycogen synthase activity was 33% greater in the athletes than in the controls (P < 0.05), and the basal glycogen synthase fractional activity was closely related to blood flow (r = 0.88, P < 0.001). In conclusion: (a) athletes are characterized by enhanced muscle blood flow and glucose uptake. (b) The cellular mechanisms of glucose uptake are increased GLUT-4 protein content, glycogen synthase activity, and glucose storage as glycogen. (c) A close correlation between glycogen synthase fractional activity and blood flow suggests that they are causally related in promoting glucose disposal.     

Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3). On day 1, pigs received an i.v. infusion of a combination of antioxidants (superoxide dismutase, catalase, and N-2 mercaptopropionyl glycine; group II, n = 9), nisoldipine (group III, n = 6), or vehicle (group I [controls], n = 9). In the control group, systolic wall thickening (WTh) in the ischemic-reperfused region on day 1 remained significantly depressed for 4 h after the 10th reperfusion, indicating myocardial "stunning." On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 53% on day 2 and 56% on day 3 compared with day 1 (P < 0.01), indicating the development of a powerful cardioprotective response (late preconditioning against stunning). In the anti-oxidant-treated group, the total deficit of WTh on day 1 was 54% less than in the control group (P < 0.01). On day 2, the total deficit of WTh was 85% greater than that observed on day 1 and similar to that observed on day 1 in the control group. On day 3, the total deficit of WTh was 58% less than that noted on day 2 (P < 0.01). In the nisoldipine-treated group, the total deficit of WTh on day 1 was 53% less than that noted in controls (P < 0.01). On days 2 and 3, the total deficit of WTh was similar to the corresponding values in the control group. These results demonstrate that: (a) in the conscious pig, antioxidant therapy completely blocks the development of late preconditioning against stunning, indicating that the production of reactive oxygen species (ROS) on day 1 is the mechanism whereby ischemia induces the protective response observed on day 2; (b) antioxidant therapy markedly attenuates myocardial stunning on day 1, indicating that ROS play an important pathogenetic role in postischemic dysfunction in the porcine heart despite the lack of xanthine oxidase; (c) although the administration of a calcium-channel antagonist (nisoldipine) is as effective as antioxidant therapy in attenuating myocardial stunning on day 1, it has no effect on late preconditioning on day 2, indicating that the ability of antioxidants to block late preconditioning is not a nonspecific result of the mitigation of postischemic dysfunction on day 1. Generation of ROS during reperfusion is generally viewed as a deleterious process. Our finding that ROS contribute to the genesis of myocardial stunning but, at the same time, trigger the development of late preconditioning against stunning supports a complex pathophysiological paradigm, in which ROS play an immediate injurious role (as mediators of stunning) followed by a useful function (as mediators of subsequent preconditioning).     

Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio.

We have examined the effects of mildly oxidized LDL and atherosclerosis on the levels of two proteins associated with HDL; apolipoprotein J (apoJ), and paraoxonase (PON). On an atherogenic diet, PON activity decreased by 52%, and apoJ levels increased 2.8-fold in fatty streak susceptible mice, C57BL/6J (BL/6), but not in fatty streak resistant mice, C3H/HeJ (C3H). Plasma PON activity was also significantly decreased, and apoJ levels were markedly increased in apolipoprotein E knockout mice on the chow diet, resulting in a 9.2-fold increase in the apoJ/PON ratio as compared to controls. Furthermore, a dramatic increase in the apoJ/PON ratio (over 100-fold) was observed in LDL receptor knockout mice when they were fed a 0.15%-cholesterol-enriched diet. Injection of mildly oxidized LDL (but not native LDL) into BL/6 mice (but not in C3H mice) on a chow diet resulted in a 59% decrease in PON activity (P < 0.01) and a 3.6-fold increase in apoJ levels (P < 0.01). When an acute phase reaction was induced in rabbits, or the rabbits were placed on an atherogenic diet, hepatic mRNA for apoJ was increased by 2.7-fold and 2.8-fold, respectively. Treatment of HepG2 cells in culture with mildly oxidized LDL (but not native LDL) resulted in reduced mRNA levels for PON (3.0-fold decrease) and increased mRNA levels for apoJ (2.0-fold increase). In normolipidemic patients with angiographically documented coronary artery disease who did not have diabetes and were not on lipid-lowering medication (n = 14), the total cholesterol/HDL cholesterol ratio was 3.1+/-0.9 as compared to 2.9+/-0.4 in the controls (n = 19). This difference was not statistically significant. In contrast, the apoJ/PON ratio was 3.0+/-0.4 in the patients compared to 0.72+/-0.2 in the controls (P < 0.009). In a subset of these normolipidemic patients (n = 5), the PON activity was low (48+/-6.6 versus 98+/-17 U/ml for controls; P < 0.009), despite similar normal HDL levels, and the HDL from these patients failed to protect against LDL oxidation in co-cultures of human artery wall cells. We conclude that: (a) mildly oxidized LDL can induce an increased apoJ/PON ratio, and (b) the apoJ/PON ratio may prove to be a better predictor of atherosclerosis than the total cholesterol/HDL cholesterol ratio.     

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA-deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death.     

大鼠局灶性脑缺血再灌注后ICAM-1表达及甲基强的松龙干预实验研究

目的:探讨细胞间粘附分子 -1(ICAM -1)在脑缺血再灌注损伤中的作用及甲基强的松龙(MP)干预后的影响。方法 :采用栓线法制备大鼠大脑中动脉(MCA)缺血再灌模型 ,运用免疫组化方法检测ICAM -1的表达 ,并观察脑组织病理学改变。结果 :ICAM -1明确表达于脑缺血再灌组手术侧半球的微血管内皮 ,MP干预后阳性微血管数减少 ,多形核白细胞浸润减少 ,组织损伤程度相对较轻。结论 :局灶性脑缺血再灌注可诱导局部脑微血管内皮细胞ICAM -1的表达 ,其表达与脑组织坏死关系密切。MP可减少ICAM -1的表达及多形核白细胞浸润。     

P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice

The expression of leukocyte and endothelial cell adhesion molecules (CAMs) is essential for the emigration of leukocytes during an inflammatory response. The importance of the inflammatory response in the development of atherosclerosis is indicated by the increased expression of adhesion molecules, proinflammatory cytokines, and growth factors in lesions and lesion-prone areas and by protection in mice deficient in various aspects of the inflammatory response. We have quantitated the effect of deficiency for intercellular adhesion molecule (ICAM)-1, P-selectin, or E-selectin on atherosclerotic lesion formation at 20 wk of age in apolipoprotein (apo) E(-/-) (deficient) mice fed a normal chow diet. All mice were apo E(-/-) and CAM(+/+) or CAM(-/-) littermates, and no differences were found in body weight or cholesterol levels among the various genotypes during the study. ICAM-1(-/-) mice had significantly less lesion area than their ICAM-1(+/+) littermates: 4.08 +/- 0.70 mm(2) for -/- males vs. 5.87 +/- 0.66 mm(2) for +/+ males, and 3.95 +/- 0. 65 mm(2) for -/- females vs. 5.59 +/- 1.131 mm(2) for +/+ females, combined P < 0.0001. An even greater reduction in lesion area was observed in P-selectin(-/-) mice: 3.06 +/- 1.04 mm(2) for -/- males vs. 5.09 +/- 1.22 mm(2) for +/+ males, and 2.85 +/- 1.26 mm(2) for -/- females compared with 5.60 +/- 1.19 mm(2) for +/+ females, combined P < 0.001. The reduction in lesion area for the E-selectin null mice, although less than that seen for ICAM-1 or P-selectin, was still significant (4.54 +/- 2.14 mm(2) for -/- males vs. 5.92 +/- 0.63 mm(2) for +/+ males, and 4.38 +/- 0.85 mm(2) for -/- females compared with 5.94 +/- 1.44 mm(2) for +/+ females, combined P < 0.01). These results, coupled with the closely controlled genetics of this study, indicate that reductions in the expression of P-selectin, ICAM-1, or E-selectin provide direct protection from atherosclerotic lesion formation in this model.     

Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion.

Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules.