Kathy Chang

Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO·) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 β-induced 1) nitrite formation (an oxidation product of NO·) and cGMP accumulation by the rat β-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ∼ 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO· in the pathogenesis of diabetic vascular complications.

Characterization and Modulation of the Immunosuppressive Phase of Sepsis

ABSTRACT Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o′)tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had ∼40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-γ) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-γ production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the hosts immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.

Inducible nitric oxide synthase (iNOS) gene deficiency increases the mortality of sepsis in mice

BACKGROUNDnNitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene knockout mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome.nnnMETHODSnC57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis.nnnRESULTSnSham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P < .01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice.nnnCONCLUSIONSnWe conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

TNF-α causes reversible in vivo systemic vascular barrier dysfunction via NO-dependent and -independent mechanisms

Tumor necrosis factor (TNF-α) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-α and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-α reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-α did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-α caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-α-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-α-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with N G-monomethly-l-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-α-treated animals. We conclude that 1) TNF-α reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.Tumor necrosis factor (TNF-alpha) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-alpha and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-alpha reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-alpha did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-alpha caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-alpha-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-alpha-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with NG-monomethly-L-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-alpha-treated animals. We conclude that 1) TNF-alpha reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.

Inhibition of Inducible Nitric Oxide Synthase Prevents Myocardial and Systemic Vascular Barrier Dysfunction During Early Cardiac Allograft Rejection

NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.

Effects of very mild versus overt diabetes on vascular haemodynamics and barrier function in rats

SummaryRats injected i. p. with a single dose of nicotinamide (250 mg/kg) 15 min prior to i.v. injection of streptozotocin (65 mg/kg) develop a very mild form of diabetes characterized by slight elevations of plasma glucose, increased levels of HbA1, and reduced insulin secretion in response to an i.v. glucose tolerance test. These rats gain weight normally and they are not hyperphagic, glycosuric, or polyuric. The effects of this very mild form of diabetes vs overt streptozotocin diabetes of three months duration on regional vascular 131I-albumin clearance, blood flow (assessed by 15 μm 85Sr-microspheres), and renal filtration function were examined in male Sprague-Dawley rats. Plasma glucose levels of rats with mild diabetes were 7.4±0.9 (mean±SD) (mmol/l) vs 6.5±0.6 for control rats and 31.3±6.0 for overtly diabetic rats. HbA1 levels were increased 1.4 fold in mildly diabetic and 2.3 fold in overtly diabetic rats. Vascular clearance of 131I-albumin was markedly increased in ocular tissues (anterior uvea, retina, and choroid), sciatic nerve, aorta, new (subcutaneous) granulation tissue, and kidney of both diabetic groups, although increases in overtly diabetic rats exceeded those in the mildly diabetic group (2.2–4.6 times control animals vs 1.6–3.3 times, respectively). Likewise, both overt and very mild diabetes markedly increased glomerular filtration rate (∼1.8 times and 1.2 times control animals, respectively), urinary excretion of endogenous albumin (∼9 times and 4 times) and IgG (∼15 times and 4 times), as well as regional blood flow in the anterior uvea, choroid, and sciatic nerve. Increases in tissue sorbitol levels were much larger in overtly diabetic rats (generally 10–20 times control animals) than in mildly diabetic rats (1.5–3 times controls). myo-Inositol levels were significantly decreased only in lens and sciatic nerve of overtly diabetic rats. These observations indicate that even very mild diabetes is associated with vascular functional changes which develop more slowly than in overtly diabetic rats, but are disproportionately large (in view of the minimal increases in glycaemia and tissue polyol levels) compared to those in overtly diabetic rats.

Albumin Permeation of New Vessels Is Increased in Diabetic Rats

125I-bovine serum albumin (BSA) permeation of the vasculature of 3-wk-old granulation tissue (induced by subcutaneous implantation of polyester fabric) formed in the diabetic milieu was assessed in female BB/W, spontaneously diabetic rats and in male, Sprague-Dawley rats with streptozocin-induced diabetes as well as in corresponding nondiabetic controls. Albumin permeation of new granulation tissue vessels was markedly increased in both groups of diabetic animals relative to that of nondiabetic controls, while albumin permeation of vessels in most other tissues did not differ for controls and diabetics. These observations indicate that the functional integrity of new vessels formed in the diabetic milieu is impaired: (1) to a greater extent than that of older vessels formed before induction of diabetes and (2) relative to new vessels in nondiabetics. The implication of these observations is that molecular constituents of vessels synthesized in the diabetic milieu are quantitatively and/or qualitatively abnormal and/or their incorporation into vessels is defective.

Tissue differences in vascular permeability changes induced by histamine

A new method is described for assessing changes in vascular permeation by albumin in multiple tissues of the same animal in response to intravascular injection of vasoactive agents. Following intravenous injection of 51Cr-RBC, 125I-BSA, and 57Co-EDTA, a test substance (i.e., histamine) is injected intravascularly or subcutaneously. Eight minutes later approximately 2.0 ml of blood is withdrawn and the heart is severed from the great vessels. Samples of tissue are then taken for determination of water content and for the ratio of counts in 125I and 51Cr in each tissue. That ratio is then divided by the corresponding ratio of the same isotopes in the blood. If the resulting quotient is greater than 1, it indicates that the volume of distribution of 125I in the tissues is greater than the ratio of plasma to red cells in large blood vessels and is indicative of permeation of the vasculature by albumin into the extravascular space. With this technique we have demonstrated that following intravenous injection of histamine, albumin permeation of vessels in the cecum is increased much more than for vessels in any other tissue in the body including skin and muscle. Following intravenous injection of 1.5 mg/kg histamine, albumin permeation in the cecum is increased 4-fold while that in skin is unchanged, except at sites where histamine also has been injected subcutaneously where it is increased 1.7-fold by 3 microgram and 10-fold by 15 micrograms of histamine. The magnitude of increases in albumin permeation of the vasculature after intravenous injection of 7.5 mg/kg of histamine was: cecum--5.1 X greater than pancreas--2.8 X greater than small intestine--2.7 X greater than cremaster and stomach--2.0 X greater than eye and aorta--1.9 X greater than fat--1.7 X greater than skin--1.6 X greater than diaphragm and forelimb--1.5 X. Even at this high dose of histamine, tissue to blood isotope ratio (tbir)-I/Cr values were not increased for heart, brain, kidney, lung, or testis. These findings attest to marked tissue differences in sensitivity to histamine-induced changes in vascular permeation by albumin. The additional that histamine-induced tbir-I/Cr increases in most tissues far exceed tbir-Co/Cr increases indicates that the increase in albumin permeation of vessels is mediated in large part by an increased rate of diffusion (rather than filtration) via an increase in the number and/or size of vascular pores large enough to accommodate albumin.

Increased susceptibility to Candida infection following cecal ligation and puncture

Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the hosts susceptibility to nosocomial infections and Candida albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem.

Tissue differences in vascular permeability induced by leukotriene B4 and prostaglandin E2 in the rat

The activity of synthetic LTB4 and PGE2, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with 125I-albumin, 51Cr-erythrocytes, and 57Co-EDTA. The rats were then injected through the carotid artery cannula with LTB4, PGE2 or a combination of LTB4 and PGE2. Eight minutes later the rats were killed and the activity of 125I, 51Cr, and 57Co measured in different organs. Changes in vascular permeability were inferred from changes in the ratios of the isotope activities. LTB4 (15 micrograms/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB4, 3 micrograms/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE2 (1 microgram/kg) enhanced vascular permeability only in the caecum. A combination of LTB4 (3 micrograms/kg) and PGE2 (1 microgram/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB4 than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB4 induced increases in permeability.