Arnold Johnson

MECHANISMS OF LUNG VASCULAR INJURY AFTER INTRAVASCULAR COAGULATION

Pulmonary intravascular coagulation and the resultant microembolization increase lung vascular permeability to proteins. The increase in permeability is mediated by the activation of cellular and humoral factors after intravascular coagulation. In particular, intravascular coagulation results in sequestration and activation of leukocytes, which appears to be of primary importance in mediating the lung vascular injury. In addition, fibrin entrapment and the generation of fibrin-degradation products after fibrinolysis also contribute to the vascular injury. The classical inflammatory agents, such as prostaglandin, histamine, bradykinin, and serotonin, may modulate the degree of injury after pulmonary vascular thrombosis, but they do not appear to be the primary mediators of the injury. Platelet aggregation did not mediate the lung vascular injury after intravascular coagulation, but was responsible for the pulmonary gas-exchange impairment that occurs with pulmonary vascular thrombosis.

Superoxide dismutase prevents the thrombin-induced increase in lung vascular permeability: role of superoxide in mediating the alterations in lung fluid balance.

We investigated the effects of superoxide dismutase (SOD) and SOD linked to Ficoil (mol. wt = 400,000) on the changes in pulmonary transvascular fluid and protein exchange following pulmonary microembolism induced with α-thrombin. Studies were made in chronically prepared unanesthetized sheep with lung lymph fistulas. Control thrombin challenged sheep (n = 5) were compared to animals infused with SOD (the SOD-thrombin group, n = 5) or animals infused with SOD linked to high molecular weight Ficoil (the Ficoll-SOD-thrombin group, n = 6). The Ficoll-SOD-thrombin animals were also compared to animals infused with Ficoil alone (the Ficoll-thrombin group, n = 4) In the control-thrombin group, thrombin induced sustained increases in the pulmonary transvascular protein clearance (pulmonary lymph flow × lymph/plasma protein concentration ratio) and pulmonary vascular resistance (PVR). In the SOD-thrombin group, thrombin initially increased both pulmonary transvascular protein clearance and PVR; however, the later increases in protein clearance and PVR were blunted. The pulmonary reflection coefficients for total protein (σ), a measure of vascular permeability to protein, decreased from a value of 0.70 ± 0.03 in normal sheep to 0.60 ± 0.01 following thrombin challenge (p < 0.05) indicating an increase in lung vascular permeability. The σ value in the SOD-treated animals was 0.70 ± 0.02, indicating a protective effect of SOD. The infusion of the Ficoll-SOD complex also attenuated the increases in pulmonary transvascular protein clearance and PVR after thrombin. However, the infusion of Ficoil alone induced a similar protection. The lymph from the SOD-thrombin and Ficoll-SOD-thrombin groups prevented the reduction of ferricytochrome C by xanthine/xanthine oxidase, whereas, the lymph from the Ficoll-thronibln animals did not have this effect, indicating SOD activity was present in the animals receiving the enzyme but not in the group infused with Ficoil alone. Differences in the degree of intravascular coagulation could not explain the response to Ficoil since the decreases in fibrinogen concentration following the thrombin were similar in all the groups. Since Ficoil and related dextrans may modify neutrophil function, in particular neutrophil adherence to the endothelium, we examined the effects of Ficoil on neutrophil adherence. The results indicated that when Ficoil was added to the endothelial medium Ficoil reduced the increase adherence of neutrophils to the endothelial cell monolayer. Therefore, Ficoil as a carrier for SOD may provide a direct protection in models of lung vascular injury that are dependent on neutrophils. The infusion of SOD prevents the thrombin-induced increase in lung vascular permeability and reduces the pulmonary vasoactive response to thrombin-induced pulmonary intravascular coagulation. The results indicate that superoxide generation contributes to the increases in lung vascular permeability to protein and PVR following thrombin.

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)

Protein oxidation as a novel biomarker of bladder decompensation

To measure the degree to which partial bladder outlet obstruction (PBOO) results in oxidative bladder damage, which subcellular components of the bladder are affected and whether these changes correlate with bladder function.

Lipoxygenase products induce neutrophil activation and increase endothelial permeability after thrombin-induced pulmonary microembolism.

We examined the mechanism of the neutrophil (PMN)-dependent increase in pulmonary vascular permeability to protein after thrombin-induced pulmonary microembolism. Humoral factors that activate PMNs after thrombin-induced pulmonary microemboUsm were characterized in pulmonary lymph obtained from unanesthetized sheep challenged with intravenous infusion of α-thrombin. Tune-dependent increases in PMN migration, aggregation, and superoxide anion (O2-) generation were induced by the puhnonary lymph obtained within 20 minutes after thrombin infusion. The puhnonary lymph neutrophil activating factors present in ether extracts of lymph had retention times of leukotriene B4 (LTB4) and monohydroxyeicosatetraenoic acids (HETEs) by high-performance liquid chromatography. The postthrombin lymph samples containing the LTB4 and HETEs increased PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Control lymph samples replete with LTB4, 5-HETE, and 15-HETE induced increases in PMN O2- generation and endothelial monolayer permeability to I25I-albumin in the presence of PMNs layered on the endothelial monolayers. Maximal increases in PMN O2-production and endothelial permeability occurred when LTB4, 5-HETE, and 15-HETE were coincubated with PMNs, indicating a synergistic action of these mediators in inducing PMN activation. Endothelial monolayer permeability to 125I-albumin did not increase with postthrombin lymph samples obtained after pretreatment with the 5-lipoxygenase inhibitor, L-651, 392. The results indicate that lipoxygenase products generated in the lungs after thrombin-induced microembolism contribute to increased endothelial permeability secondary to PMN activation.

Fibrin degradation products increase lung transvascular fluid filtration after thrombin-induced pulmonary microembolism

The effect of fibrin degradation products (FDP) on pulmonary transvascular fluid and protein exchange was examined in the sheep lung lymph fistula preparation. The pulmonary lymph was used to assess changes in pulmonary lymph flow (Qlym) (a measure of net transvascular fluid filtration rate) and the lymph/plasma protein concentration ratio (L/P) (a measure of protein seiving across the microvascular barrier). Studies were made in 3 groups: Control-Thrombin (n = 7) received 96.9 +/- 9.4 U/kg of alpha-thrombin, Control-FDP (n = 6) received infusion of FDP prepared by plasmin digestion of fibrin, and Thrombin-FDP (n = 5) received thrombin (102.0 +/- 7.5 U/kg) and then an FDP infusion was begun at 60 min after the thrombin. In the Control-Thrombin animals, Qlym increased without a change in the L/P after thrombin, indicating an increase in vascular permeability to proteins. In the Control-FDP group, infusion of FDP had no effect on Qlym and L/P. In the Thrombin-FDP group, thrombin increased Qlym with no change in the L/P and the subsequent infusion of FDP further increased Qlym but slightly decreased the L/P, an effect not seen in the Control-FDR group. The results indicate that FDP infusion alone does not alter pulmonary transvascular fluid and protein exchange. However, in the presence of thrombin-induced pulmonary microembolization FDP infusion enhances the net transvascular fluid filtration rate, probably by increasing the capillary hydrostatic pressure.

TNF induced activation of pulmonary microvessel endothelial cells: a role for GSK3β

The hypothesis tested was PKCalpha mediates the phosphorylation of glycogen synthetase kinase 3beta (GSK3beta) and that the GSK3beta inhibition modulates the response to tumor necrosis factor-alpha (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKCalpha in the phosphorylation of GSK3beta (i.e., an indicator of GSK3beta inhibition), PMEC were pretreated with 1) nonsense-RNA-PKCalpha, 2) siRNA-PKCalpha, and 3) the PKC inhibitor Gö6983. In the nonsense RNA-PKCalpha+TNF and TNF groups, there was increased phosphorylated GSK3beta-Ser9 that did not occur in the Gö6983+TNF group. In the TNF groups, there was a significant correlation between PKCalpha protein and phosphorylated GSK3beta-Ser9 that did not occur in the groups without TNF. Second, to assess the role of GSK3beta in beta-catenin activity, PMEC were pretreated with 1) wild-type (w) GSK3beta plasmid to enhance GSK3beta activity, 2) kinase dead (kd)-GSK3beta plasmid, and 3) the GSK3beta inhibitor SB-216763. In the TNF group, there was increased unphosphorylated beta-catenin-Ser37/33 compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a beta-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKCalpha-dependent inhibition of GSK3beta.

The effect of chronic partial bladder outlet obstruction on corpus cavernosum smooth muscle and Rho-kinase in rabbits.

The goal of this study was to investigate the effect of different severities in bladder dysfunction on corpus cavernosum physiology, morphology and expression of Rho‐kinase in rabbits.

Lipoteichoic Acid from Staphylococcus aureus Induces Lung Endothelial Cell Barrier Dysfunction: Role of Reactive Oxygen and Nitrogen Species

Tunneled central venous catheters (TCVCs) are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus) biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA), a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2). The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM) that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam(3)CSK(4) induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS) activation (as measured by the p-eNOSser1177:p-eNOSthr495 ratio). The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.

Tumor necrosis factor-α induces increased lung vascular permeability: A role for GSK3α/β

We tested the hypothesis that glycogen synthase kinase 3α/β (GSK3α/β) modulates tumor necrosis factor-a (TNF) induced increased lung vascular permeability. Rats were treated with TNF (i.v., ~100ng/ml) or vehicle 0.5h, 4.0h and 24.0h prior to lung isolation. Rats were co-treated with the GSK3α/β inhibitors SB216763 (0.6mg/kg) or TDZD-8 (1.0mg/kg). After TNF, the isolated lung was assessed for hemodynamics, wet-dry/dry weight (W-D/D) and extravascular albumin. Extravascular albumin significantly increased at TNF-24h compared to Control. In the GSK3α/β-inhibited+TNF groups, extravascular albumin was similar to the Control and respective SB216763 and TDZD-8 groups. In separate studies, to assess GSK3α/β-activity, lung lysate was assessed for phospho-GSK3α/β-Ser(21/9), total GSK3α/β, un-phospho-β-catenin-Ser(33/37) and total β-catenin. In the TNF-4.0h group, there was no change in GSK3α/phospho-GSK3α-Ser(21) but there was an increase in GSK3β/GSK3β-Ser(9) compared to Control, indicating GSK3β activation at TNF-4.0h. GSK3β activation was verified because there was a decrease in un-phospho-β-catenin-Ser(33/37)/β-catenin in the TNF-4.0 group, a specific outcome for GSK3β activation. In the SB216763+TNF group, un-phospho-β-catenin-Ser(33/37) was similar to Control, indicating prevention of TNF-induced GSK3β activation. In the TNF-24h group, there were increases in the biomarkers of inflammation phospho-eNOS-Ser (1117) and oxidized protein, which did not occur in the SB216763+TNF-24h and TDZD-8+TNF-24h groups. In the SB216763+TNF-24h and TDZD-8+TNF-24h groups, un-phospho-β-catenin-Ser(33/37) was greater than in the Control, indicating continued inhibition of GSK3β. The data indicates that pharmacologic inhibition of GSK3β inhibits TNF induced increased endothelial permeability associated with lung inflammation.