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Journal of Lipid Research, Vol. 47, 2148-2160, October 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
* Department of Medicine, David Geffen School of Medicine at the University of California–Los Angeles, Los Angeles, CA 90095-1679
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at the University of California–Los Angeles, Los Angeles, CA 90095-1679
The Atherosclerosis Research Unit, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294
Published, JLR Papers in Press, July 12, 2006.
1 To whom correspondence should be addressed. e-mail: gbuga{at}mednet.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(P < 0.01) and CCL2/monocyte chemoattractant protein-1 (P < 0.001). A WD increased (P < 0.001) brain arteriole wall thickness and smooth muscle -actin, which was reduced by D-4F but not by ScD-4F (P < 0.0001). There was no difference in plasma lipid levels, blood pressure, or arteriole lumen diameter with D-4F treatment. Cognitive performance in the T-maze continuous alternation task and in the Morris Water Maze was impaired by a WD and was significantly improved with D-4F but not ScD-4F (P < 0.05). We conclude that a WD induces brain arteriole inflammation and cognitive impairment that is ameliorated by oral D-4F without altering plasma lipids, blood pressure, or arteriole lumen size.
Supplementary key words hyperlipidemia • lipoproteins • apolipoprotein A-I mimetic peptides • arterioles • hyperlipidemia • brain function
Abbreviations: LDLR–/–, LDL receptor-null; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; MWM, Morris Water Maze; ScD-4F, scrambled D-4F; T-CAT, T-maze continuous alternation task; WD, Western diet
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pathologic effects of hyperlipidemia on small vessels have focused on measurements of vasoreactivity. Ou et al. (3) noted that there was increased wall thickness of the facialis artery after feeding a WD to LDLR–/– mice and that oral D-4F prevented and reversed this thickening. In vitro collagen synthesis was stimulated by oxidized sterols, and this was prevented by treatment with D-4F. Ou et al. (3) implied that the increased collagen synthesis was likely to be responsible in part for the in vivo thickening of the arterial wall.
The accumulation of macrophages in the subendothelial space of large arteries in response to LDL-induced chemokines would be expected, because i) LDL is trapped in the subendothelial space and ii) a few sentinel macrophages are always present in this space as part of the innate immune system (4–6). The work of Napoli et al. (5) indicates that in large arteries such as the aorta, macrophages are active in the subendothelial space, even in the human fetus.
The facialis artery studied by Ou et al. (3) has a lumen diameter of 180–280 µm, and there is no evidence to suggest that macrophages accumulate in the subendothelial space of such small arteries. Arterioles have lumen diameters of 10 to 100 µm, and there is no evidence of a significant subendothelial space in these small vessels. It is not likely that macrophages could accumulate between the monolayer of luminal endothelial cells and the smooth muscle cell layer that surrounds them without obstructing the lumen. Indeed, in arterioles, sentinel macrophages have been shown to reside not in the subendothelial space as in large arteries, but rather on the adventitial side of the vessel (7, 8).
Mouse models suggest that diseases such as Alzheimer's disease may have an inflammatory component similar to atherosclerosis that is associated with very small vessels such as arterioles (9–11). In humans, microvessel-associated monocyte chemoattractant protein-1 (MCP-1) and interleukin-1ß were found in Alzheimer's brains but not in non-Alzheimer's brains (12).
The role of hyperlipidemia in causing inflammation of very small vessels such as brain arterioles has not been previously studied. We report here that feeding a WD to LDLR–/– mice induced brain arteriole inflammation and cognitive impairment, which were ameliorated by oral D-4F without altering plasma lipids, blood pressure, or arteriole lumen size.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice
Female wild-type and LDLR–/– C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were maintained on a chow diet (Ralston Purina) prior to administration of a WD (Teklad/Harlan, Madison, WI; diet No. 88137, 42% fat, 0.15% cholesterol, w/w). The WD was administered to the mice when they were 5–6 weeks old, and the diet was continued for 6–8 weeks. At the time that the WD was started, D-4F or ScD-4F was added to the drinking water at 300 µg/ml and provided ad libitum for 6–8 weeks prior to functional testing and histologic examination. In pilot studies, this period of feeding the WD was found sufficient to produce significant changes in brain arterioles and in cognitive behavior. The mice consumed approximately 2.5 ml of water per day per mouse, and there was no significant difference in water or food consumption between groups. The University of California–Los Angeles Animal Research Committee approved all studies.
Histopathology
For studies of brain arteriole wall thickness, brains were perfusion fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4 at physiologic pressures (75–85 mm/Hg for the anesthetized mice) as described by Fernagut et al. (14). Brains were cryopreserved in 10% sucrose in PBS (pH 7.4), embedded in OCT (Tissue-Tek; Miles Laboratories, Ltd., Elkhart, IN), frozen in isopentane at –40°C, and sectioned in a cryostat at –20°C. The frozen brains were coronally cut into 8 µm sections and stained with hematoxylin-eosin (H and E) or immunostained for smooth muscle -actin (Spring Bioscience, Fremont, CA). Paraffin embedding was also used at the beginning of the study to compare with frozen brain sections. Because comparable results were obtained, both the histological and immunohistochemical studies were performed on frozen sections. Immunohistochemistry for brain macrophage/microglia, CCL3/macrophage inflammatory protein-1 (MIP-1), and MCP-1 was performed on unperfused freshly removed brains frozen in isopentane at –40°C, rapidly embedded in OCT, and coronally cut into 8 µm sections in a cryostat at –20°C. The following primary antibodies and dilutions were used: rabbit anti-mouse -actin polyclonal antibody (1:100; Spring Bioscience); rat anti-mouse F4/80 (1:250; Serotec, Raleigh, NC); goat polyclonal anti-MIP-1 (1:25; Santa Cruz Biotechnology, Santa Cruz, CA); and goat polyclonal anti-MCP-1 antibody (1:50; Santa Cruz Biotechnology). The following secondary antibodies and dilutions were used: goat anti-rabbit (1:200, Jackson Immuno Research Labs, Inc., West Grove, PA); goat anti-rat (1:200, Jackson Immuno Research Labs); and donkey anti-goat and donkey anti-rabbit (1:500, Santa Cruz Biotechnology). Vessels were studied in coronal sections from throughout the brain, and all serial sections were used for measurements. Staining for F4/80, MIP-1, and MCP-1 was performed on serial sections less than 10 µm apart to determine whether the two antibodies localized to the same arteriole. Controls for the immunostaining consisted of sections exposed to secondary-only antibodies in addition to IgG (Sigma-Aldrich, Dallas, TX) and rabbit anti-mouse actin antibody in the case of MCP-1 and F4/80 or to blocking peptide in the case of MIP-1 (Santa Cruz Biotechnology).
Morphometry and associated statistical methods
Measurements of vascular wall thickness were made only on arterioles that were fully distended and perpendicularly cross sectioned. Using a 40x microscope objective, the sectioned vessels were photographed using SPOT Image software. Three measurements of the internal and external diameters were taken for each and were averaged. Vessel sizes were between 10 and 160 µm, and the comparison of wall-to-lumen ratios was made separately for arterioles with internal diameters of 10–20 µm, 21–50 µm, and 51–100 µm. A minimum of 20 arterioles from each group were examined in the cortical area and in the deep white-matter regions from each brain, and the wall thickness and wall-to-lumen ratios were determined. The ratio of immunoreactive wall thickness to the internal diameter of each vessel was determined in sections immunostained for smooth muscle -actin. All measurements were performed on a single focal plane by one investigator and repeated by two observers blinded to treatment, using an Olympus BH-2 microscope equipped with a 40x lens. The coefficient of variation for interobserver measurements was found to be 14 ± 1%. The total number of brain arterioles and the number of arterioles demonstrating positive F4/80, MIP1-, or MCP-1 immunostaining were counted to determine the percent of immunolabeling. Twenty microscope fields from each brain were counted at a final magnification of 200x by one investigator and repeated by two observers blinded to treatment. The coefficient of variation for interobserver measurements was found to be 10 ± 1%. All nonvascular cells (those immunopositive for MCP-1 or MIP-1 and those immunonegative) situated within a circle with a diameter of 150 µm around each arteriole were counted at a final magnification of 400x, and the percent of immunopositive cells was determined. Statistical calculations for morphometry were performed using InStat software (Graph Pad, San Diego, CA).
Behavioral studies and associated statistical methods
T-maze continuous alternation task (T-CAT) testing was performed by one investigator unaware of the treatment groups. The T-maze apparatus and the T-CAT procedure were identical to those described by Gerlai et al. (15). T-CAT consisted of 1 forced and 14 free-choice trials. Consecutive choices made by the mice were counted, and the number of alternations and the percent of alternations different from chance during the 14 free-choice trials were calculated (0%, no alternation; 100%, alternation at each trial; 50%, chance level). The T-maze apparatus, separated from the investigator by a black curtain, was operated by remote control, and the undisturbed movement of the mice in the maze was observed on a television monitor and videotaped. Statistics were performed using StatView software (SAS Institute, Cary, NC).
The hidden-platform version of the Morris Water Maze (MWM) task (9, 11, 16) was used to assess spatial learning and orientation by LDLR–/– mice on a WD with 300 µg/ml D-4F versus 300 µg/ml of ScD-4F in drinking water. Mice were given a total of 12 training trials (4 trials per day for 3 days), followed by 4 acquisition trails per day for 3 or 4 days, with their performance continuously registered by a video tracking system (SD Instruments, Inc., San Diego, CA). Statistics were performed using StatView software (SAS Institute).
Other procedures
Plasma lipoprotein and lipid levels were determined as described previously (13, 17). Blood pressure was determined by the tail cuff method (18, 19). Electron microscopy was performed as previously described (20).
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RESULTS |
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associated with brain arterioles
The association of MIP-1 with brain arterioles was determined by immunostaining (Fig. 2A
). The percent of brain arterioles with MIP-1 immunostaining was significantly increased in LDLR–/– mice fed a WD and given the control inactive peptide ScD-4F compared with mice on chow or on a WD with D-4F in their drinking water (Fig. 2B). The distribution of F4/80 immunostaining associated with the brain arterioles was different from that of MIP-1 (Fig. 2C), in that the latter was more broadly distributed, consistent with its role as a secreted chemokine (21) and its known ability to bind to vascular smooth muscle cells (22).
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To exclude the possibility that dissimilar-sized vessels were compared in the two experimental groups (D-4F versus ScD-4F), which might have accounted for the difference in wall thickness, we measured the internal diameters of all arterioles used for morphometry. There was no significant difference in the lumen size of brain arterioles between the mice receiving D-4F or ScD-4F (Fig. 4C).
Some investigators have suggested that the most reliable measurement of arteriole wall thickness is obtained by dividing the wall thickness for each arteriole by the lumen diameter for that arteriole (23). Figure 4D demonstrates that in 21–50 µm arterioles, the wall-to-lumen ratio was significantly less in mice receiving D-4F compared with the control inactive peptide ScD-4F. Significant reductions were also noted in brain arterioles with lumen diameters of 10–20 µm and 51–100 µm (data not shown).
There was a positive correlation between the number of microglia associated with brain arterioles and the thickness of the wall of the arteriole (r = 0.6513 and r2 = 0.4241; P < 0.0001). Thus, wall thickness increased in direct proportion to the number of microglia associated with the arterioles. This positive association was not surprising, because as noted above, on electron microscopy, many of the microglia were found incorporated within the basal lamina of the arteriole. However, although highly significant, the correlation suggests that other factors were also involved in causing the increased wall thickness.
D-4F decreases hyperlipidemia-induced brain arteriole smooth muscle -actin
Perfusion-fixed brains were immunostained for smooth muscle -actin (Fig. 5A
). Administration of a WD to LDLR–/– mice resulted in a significant increase in the media-to-lumen ratio as determined from sections immunostained for smooth muscle -actin (Fig. 5B). In brain arterioles with lumen diameters of 21–50 µm, treatment with D-4F significantly reduced the media-to-lumen ratio compared with mice treated with the control inactive peptide ScD-4F in sections immunostained for smooth muscle -actin (Fig. 5C). Similar reductions of the media-to-lumen ratio were noted in brain arterioles stained for smooth muscle -actin with lumen diameters of 10–20 µm and 51–100 µm (data not shown). It was not possible to determine whether the increase in smooth muscle -actin was due to an increase in smooth muscle cell numbers or increased content of smooth muscle cell -actin in each smooth muscle cell or some combination of the two. Nor was it possible to quantify how much of the increased wall thickness was due to an increase in extracellular matrix.
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T-CAT When placed in a T-maze, normal mice will, more often than chance, remember on successive trials that the arm of the T-maze that they entered on the previous trial was a "blind alley" and normal mice will "alternate" on the successive trial to the opposite arm of the T-maze. This process, which involves working memory, is called "spontaneous alternation." In contrast to normal mice, mice with impaired cognition will, more often than chance, enter the blind alley again on the successive trial. Thus, impaired mice will show a significant decrease in the number of spontaneous alternations. There was a significant decrease in the number of alternations in LDLR–/– mice on a WD compared with chow (Fig. 6A ). On chow, LDLR–/– mice, more often than chance, demonstrated spontaneous alternations, but on a WD, more often than chance, the mice demonstrated impaired behavior (i.e., more often than chance, they returned to the blind alley) (Fig. 6B). Treatment with D-4F (but not the control inactive peptide ScD-4F) improved their working memory, as measured by a significant increase in the number of spontaneous alternations and the percent of alternations different from chance (i.e., different from 50%) (Fig. 6C, D).
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D-4F did not alter blood pressure or plasma lipids
Trieu and Uckun (19) reported that feeding a WD to male (but not female) LDLR–/– mice resulted in increased blood pressure. Because our mice were female, we would not have expected the WD to alter their blood pressure. Indeed, this was the case, and D-4F also did not alter blood pressure (data not shown).
There was no significant difference in the concentrations of plasma total cholesterol, LDL+VLDL-cholesterol, HDL-cholesterol, or triglycerides when the mice were administered D-4F compared with the control inactive peptide ScD-4F (Table 1 ).
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expression in nonvascular brain cells, which were produced by the arterioles in response to the WD (Figs. 2, 3), may have acted on adjacent nonvascular brain cells. Chemokines have been shown to be synthesized by all major cell types in the brain, i.e., neurons, glia, microglia, and all major cell types in the brain have the receptors for these chemokines (25, 26). Therefore, we determined the percent of nonvascular cells immunopositive for MCP-1 and MIP-1 within 150 µm surrounding each brain arteriole (Fig. 7
). The percent of nonvascular cells that were immunopositive for MCP-1 (Fig. 7A, B) and MIP-1 (Fig. 7C) in LDLR–/– mice on a WD with the control inactive peptide ScD-4F in the drinking water was significantly greater compared with mice on chow or on a WD with D-4F in the drinking water. The total number of nonvascular cells in the area surrounding each brain arteriole was not different between groups (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and MCP-1). However, the location of the microglia was quite different in brain arterioles compared with the location of macrophage cells in larger arterial vessels. The microglia were always located on the adventitial side of the arteriole, never between the smooth muscle cells and the endothelial cells. Often the microglia were incorporated within the basal lamina of the arteriole, indicating the intimate nature of the association. The positive correlation between the number of microglia per arteriole and wall thickness indicates that the number of microglia per arteriole directly contributed to wall thickness (i.e., the incorporation of some microglia within the basal lamina of the arteriole wall directly contributed to the measurements of wall thickness as measured in H and E-stained sections). However, the correlation (r2 = 0.4241; P < 0.0001), although highly significant, also suggests that other factors must have been involved in determining wall thickness differences between the treatment groups. Wall thickness increases with increasing vessel size (measured by the internal diameter of the vessel), and so it is critical to compare vessels of comparable size. Therefore, all of our studies compared vessels of similar size from the different treatment groups, as stated in the text and as illustrated by the data in Fig. 4C.
Bush et al. (27) have emphasized the importance of monocyte-macrophages and MCP-1 in vascular hypertrophy. Perhaps the microglia associated with brain arterioles in LDLR–/– mice on a WD also indirectly contributed to the increase in brain arteriole smooth muscle -actin via the secretion of MCP-1.
We chose to study the two CC (ß-chemokine) family members CCL2/MCP-1 and CCL3/MIP-1 because they are both frequently secreted during wound healing and following neurotrauma. For example, after target ablation by olfactory bulbectomy, there was synchronized degenerative cell death of olfactory neurons followed by infiltration of macrophages that was mediated by MIP-1 and MCP-1 (28). After deletion of mitogen-activated protein kinase-activated protein kinase 2 in mice, the release of both of these chemokines was inhibited from bacterial lipopolysaccharide and interferon-
-stimulated microglial cells (21). MCP-1 is secreted by cytokine-stimulated vascular smooth muscle cells (29), as is the case for cytokine-stimulated endothelial cells (30). Additionally, MIP-1 and MCP-1 are both found in lesions of mouse models of atherosclerosis (31).
The LDL-mediated induction of chemokines by human artery wall cells is mediated by oxidized lipids that are removed or inactivated by apoA-I, normal HDL, and the antioxidant enzyme associated with HDL, paraoxonase (32, 33). In addition to contributing to LDL induction of chemokine production by artery wall cells, these oxidized lipids inhibit some of the antioxidant enzymes associated with HDL (34). D-4F is an apoA-I mimetic peptide that removes these oxidized lipids from lipoproteins (34). As a result of the removal of these oxidized lipids, the antioxidant enzymes associated with HDL are activated and there is a further reduction in the proinflammatory lipids (34). As a result, D-4F significantly reduces lesions in mouse models of atherosclerosis (35) and reduces inflammation after viral infection (36, 37). Although we did not measure these parameters in this study, these properties of D-4F presumably contributed to the beneficial effects demonstrated here.
The increase in the percent of brain arterioles with associated macrophages (Fig. 1) was approximately 2-fold on the WD. Although the increases in MIP-1 (Fig. 2) and MCP-1 (Fig. 3) were highly significant, they were less than 2-fold. This discrepancy may suggest that other as yet unidentified chemokines may play an important role in hyperlipidemia-induced arteriole inflammation.
We chose to use only female mice in these studies because of their relative behavioral placidity and because they have previously been found to be immune from the increase in blood pressure that follows feeding the WD to male LDLR–/– mice (19). Our results confirmed those of Trieu and Uckun (19) in that we did not find any change in blood pressure with administration of the WD in these female mice, nor did we see a change in blood pressure with administration of either D-4F or the inactive control peptide ScD-4F.
The results presented here, together with those previously reported (1–3), indicate that a WD in LDLR–/– mice induces an inflammatory response throughout the arterial tree. In arterioles, the macrophages are located on the adventitial side of the vessel (7, 8) in close proximity to nonvascular cells. As reported here, by electron microscopy, many of the microglia were incorporated into the wall of the arteriole within the basal lamina. Because apoB-containing lipoproteins do not cross the blood-brain barrier, the improvement of cognitive behavior in the T-CAT and MWM tests (Fig. 6) after D-4F treatment may be due, at least in part, to the reduction in the inflammatory response (as assessed by MIP-1 and MCP-1 immunostaining) of nonvascular cells adjacent to brain arterioles in the D-4F-treated mice (Fig. 7). Chemokines are produced by all major cell types in the brain, and the major cell types in the brain have the receptors for these chemokines, suggesting that chemokine signaling in the brain probably has functions beyond a role in neuroinflammation (e.g., chemokine receptors expressed by mature neurons may regulate synaptic transmission and neuronal survival) (25, 26).
It has long been assumed that tissue dysfunction induced by hyperlipidemia in the absence of obstruction of the vascular lumen was due to abnormalities in vascular reactivity that may alter blood flow to the tissues. We did not measure cerebral blood flow in the current studies. It is entirely possible that alterations in cerebral blood flow due to alterations in vasoreactivity may have contributed to the observed changes in cognitive function. However, the data presented here suggest that such changes may also be due, in part, to the spread of chemokines from brain arterioles to adjacent nonvascular brain cells (Fig. 7). The data presented here, although consistent with this hypothesis, do not conclusively establish that the changes in cognitive function were due to inflammatory chemokines radiating from brain arterioles. It is possible that the WD induced a generalized inflammation of the brain that was independent of arteriole inflammation. For example, it may be that a generalized increase in circulating cytokines was induced by the WD and that these cytokines acted on the brain cells directly. We did not measure circulating cytokines in these studies. D-4F is known to be associated with HDL (34), and we do not know whether D-4F crosses the blood-brain barrier. We do know that D-4F can reduce the levels of circulating cytokines (36), and this action appears to be related to its ability to reduce proinflammatory oxidized lipids (37).
Speaking against the possibility that the WD induced a generalized inflammation of the brain that was mitigated by D-4F was the finding that upon feeding the WD, F4/80 immunostaining only increased in association with arterioles. Moreover, F4/80 immunostaining was only reduced by D-4F in this location. Upon feeding the WD, there was no increase in F4/80 immunostaining of the brain that was not intimately associated with the arterioles. Moreover, F4/80 immunostaining of the brain in areas removed from the arterioles was not affected by any of the treatments. Therefore, we believe that the concept of arterioles as centers of hyperlipidemia-induced inflammation from which soluble chemokines can spread to adjacent nonvascular cells and induce dysfunction is novel and deserving of further study.
The ability of oral D-4F to decrease the WD-induced inflammation associated with brain arterioles and the ability of D-4F to ameliorate the WD-induced deterioration in cognitive performance without altering blood pressure, plasma lipid, or lipoprotein levels suggests that apoA-I and apoA-I mimetic peptides may have therapeutic potential in a variety of diseases of small arterial vessels.
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ACKNOWLEDGMENTS |
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Manuscript received May 15, 2006 and in revised form June 27, 2006.
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REFERENCES |
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and monocyte chemoattractant protein-1. J. Neurosci. Res. 70: 784–793.[CrossRef][Medline]. Circ. Res. 93: 515–522.
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