No abstract

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.

Increased permeability of the blood-brain barrier (BBB) is important in neurological disorders. Neuroinflammation is associated with increased BBB breakdown and brain injury. Tumor necrosis factor (TNF)-␣ is involved in BBB injury and edema formation through a mechanism involving matrix metalloproteinase (MMP) up-regulation. There is emerging evidence indicating that cyclooxygenase (COX) inhibition limits BBB disruption following ischemic stroke and bacterial meningitis, but the mechanisms involved are not known. We used intracerebral injection of TNF-␣ to study the effect of COX inhibition on TNF-␣-induced BBB breakdown, MMP expression/activity, and oxidative stress. BBB disruption was evaluated by the uptake of 14 C-sucrose into the brain and by magnetic resonance imaging utilizing gadolinium-diethylenetriaminepentaacetic acid as a paramagnetic contrast agent. Using selective inhibitors of each COX isoform, we found that COX-1 activity is more important than COX-2 in BBB opening. TNF-␣ induced a significant up-regulation of gelatinase B (MMP-9), stromelysin-1 (MMP-3), and COX-2. In addition, TNF-␣ significantly depleted glutathione as compared with saline. Indomethacin (10 mg/kg i.p.), an inhibitor of COX-1 and COX-2, reduced BBB damage at 24 h. Indomethacin significantly attenuated MMP-9 and MMP-3 expression and activation and prevented the loss of endogenous radical scavenging capacity following intracerebral injection of TNF-␣. Our results show for the first time that BBB disruption during neuroinflammation can be significantly reduced by administration of COX inhibitors. Modulation of COX in brain injury by COX inhibitors or agents modulating prostaglandin E 2 formation/signaling may be useful in clinical settings associated with BBB disruption.

Proteolytic disruption of the extracellular matrix with opening of the blood-brain barrier (BBB) because of matrix metalloproteinases (MMPs) occurs in reperfusion injury after stroke. Matrix metalloproteinase inhibition blocks the early disruption of the BBB, but the long-term consequences of short-term MMP inhibition are not known. Recently, a method to quantify BBB permeability by graphical methods was described, which provides a way to study both early disruption of the BBB and long-term effects on recovery in the same animal. We used a broad-spectrum MMP inhibitor, BB1101, to determine both the usefulness of the Magnetic resonance imaging (MRI) method for treatment studies and the long-term effects on recovery. Magnetic resonance imaging studies were performed in control (N = 6) and drug-treated (N = 8) groups on a dedicated 4.7-T MRI scanner. Adult Wistar-Kyoto underwent a 2-h middle cerebral artery occlusion followed by an MRI study after 3 h of reperfusion, which consisted of T2-and diffusion-weighted techniques. Additionally, a rapid T1 mapping protocol was also implemented to acquire one pre-gadolinium-diethylenetriaminepentaacetic acid baseline data set followed by postinjection data sets at 3-min intervals for 45 mins. The same animal was imaged again at 48 h for lesion size estimation. Data was postprocessed pixel-wise to generate apparent diffusion coefficient and permeability coefficient maps. Treatment with BB-1101 significantly reduced BBB permeability at 3 h, but failed to reduce lesion size at 48 h. Behavioral studies showed impairment in recovery in treated rats. Magnetic resonance imaging allowed for the monitoring of multiple parameters in the same animal. Our studies showed that BB-1101 was an excellent inhibitor of the BBB damage. However, results show that BB-1101 may be responsible for significant deterioration in neurologic status of treated animals. Although these preliminary results suggest that BB-1101 is useful in reducing early BBB leakage owing to reperfusion injury in stroke, further studies will be needed to determine whether the later detrimental effects can be eliminated by shorter time course of drug delivery.

Abbreviations used: BBB, blood-brain barrier; Gd-DTPA, gadolinium-diethylenetriamine pentaacetic acid; MCAO, middle cerebral artery occlusion; MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; NBO, normobaric hyperoxia; PBS, phosphate-buffered saline; TTC, 2,3,5-triphenyltetrazolium chloride. AbstractMatrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O 2 ) or normoxia (21% O 2 ) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91 phox and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91 phox expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91 phox containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.

Electron paramagnetic resonance imaging (EPRI) is a new modality for visualizing O2 distribution in tissues, such as the brain following stroke or after administration of drugs of abuse. We have recently shown that 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] is a pro-imaging agent that can cross the blood-brain barrier. After hydrolysis by esterases, the anion of 3-carboxy-2,2,5,5-tetramethyl-1-tetramethyl-1-pyrrolidinyloxyl [2] is trapped in brain tissue. In the present study, we investigated the feasibility of using this to map the changes of O2 concentration in mouse brain following focal ischemia. The decrease in tissue O2 concentration in the ischemic region of mouse brain was clearly visualized by EPRI. The hypoxic zone mapped by EPRI was spatially well-correlated with the infarction area in the brain imaged by diffusion-weighted MRI. Finally, we observed a decrease in the size of the hypoxic region, when the mouse breathed higher levels of O2. This finding suggests that EPRI with specifically designed nitroxides is a promising imaging modality for visualizing O2 distribution in brain tissue, especially in an ischemic brain. We believe that this imaging method can be used for monitoring the effects of therapeutic intervention, aimed at enhancing brain O2 supply, which is crucial in minimizing brain injury following stroke.

White matter (WM) injury after bilateral common carotid artery occlusion (BCAO) in rat is associated with disruption of the blood-brain barrier (BBB) by matrix metalloproteinases (MMPs). We hypothesized that WM injury as seen on magnetic resonance imaging (MRI) would correlate with regions of increased MMP activity. MRI was performed 3 days after BCAO surgery in rats. Apparent diffusion coefficients (ADC) were calculated and vascular permeability was quantified by the multiple-time graphical analysis (MTGA) method, using gadolinium-diethylenetriamine pentaacid (Gd-DTPA). After MRI, one group of animals had BBB permeability measured in the WM with (14)C-sucrose, and another had Evans blue (EB) injected for fluorescent microscopy for MMP-2, MMP-9, tight junction proteins (TJPs), and in situ zymography. We found that ADC values were increased in WM in BCAO rats compared with controls (P<0.05). WM with increased ADC had leakage of EB. MMP-2 and MMP-9 activity on in situ zymograms corresponded with leakage of EB. Although increased permeability to EB could be visualized, permeability quantification with (14)C-sucrose and Gd-DTPA failed to show increases and TJPs were intact. We propose that increased ADC, which is a marker of vasogenic edema, is related to activity of MMP-2 and MMP-9. MRI provides unique information that can be used to guide tissue studies of WM injury.