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【关键词】 macrophage
Division of Nephrology and Renal Research Institute, Departments of 1Medicine, 2Cell Biology
3Pathology, New York Medical College, Valhalla, New York
ABSTRACT
Interactions of leukocytes with the vascular endothelium culminating in their diapedesis represent not only a crucial event in immune surveillance and defense but are also critically involved in the pathogenesis of many inflammatory diseases, including atherosclerosis. Our previous in vitro studies using atomic force microscopy measurement of monocyte-endothelial cell interaction have demonstrated that a cyclic arginine-glycine-aspartic acid peptide (cRGD) inhibited their adhesion through very late antigen (41-integrin; VLA4)-vascular cell adhesion molecule-1 by 60% with the IC50 = 100 nM. To elucidate the potential efficacy of this peptide in vivo in preventing atherogenesis, experiments were performed in apolipoprotein E (ApoE)-deficient (/) mice fed a Western diet and receiving chronic treatment with cRGD peptide for 24 wk. In addition, some animals were subjected to a temporary carotid artery ligation while receiving the above treatment. Formation of fatty streaks and infiltration of the vascular wall with macrophages were not affected by cRGD treatment. Infiltration of the carotid artery postligation was significantly reduced in the cRGD-treated animals, as was the lipid accumulation. Furthermore, cRGD-treated ApoE/ mice exhibited significantly lesser macrophage infiltration and lipid accumulation in the kidneys, the site of the highest expression of VLA4. These data demonstrated that cRGD peptide is a potent inhibitor of monocyte/macrophage infiltration of the injured macrovasculature and of the renal microvasculature, where it results in the attenuation of lipid accumulation. Formation of fatty streaks in the aortic root was not inhibitable by this treatment.
atherosclerosis; apolipoprotein E; VLA-4; kidney; lipid accumulation
INTERACTIONS OF LEUKOCYTES with the vascular endothelium culminating in their diapedesis represent not only a crucial event in immune surveillance and defense but are also critically involved in the pathogenesis of many inflammatory diseases, including atherosclerosis. The process of leukocyte egress has been tentatively subdivided into stages of rolling, cell activation, firm adhesion, and transendothelial migration (TEM), with each stage engaging a distinct set of adhesion molecules and their activation (5, 13, 26). These adhesion molecules include L-selectin; carbohydrate ligands of selectins; 1-, 2-, and 7-integrins; and platelet endothelial cell adhesion molecule (PECAM)-1 on leukocytes, and P- and E-selectins, intercellular adhesion molecule (ICAM)-1 and ICAM-2, vascular cell adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule-1 (MadCAM-1), PECAM-1 and 1-integrins on endothelial cells (31). Therapeutic strategies based on inhibiting some of these interactions with neutralizing antibodies have been applied to the animal models of ischemia-reperfusion and acute inflammatory processes with variable success (15, 30). The role of monocyte/macrophage infiltration of the intima of the arterial wall in the pathogenesis of atherosclerotic plaques is well recognized (17). The end effect of the inflammatory infiltrate largely depends on the "floodgate" of the infiltrating cells, their adhesion to the endothelium and TEM, both dictating the rate of release of their cargo [myeloperoxidase, hydrogen peroxide, hypohalates, chloramines, reactive nitrogen intermediates, leukotriens and platelet-activating factor, elastase, collagenase, gelatinase, serine protease, etc. (reviewed in Ref. 20)]. Leukocyte docking and TEM require, in addition to L-selectins, leukocyte-activating chemotactic factors and leukocyte integrins, active participation of endothelial cells with their P- and E-selectins, VCAM-1, ICAMs, and MadCAM (28). Integrins and other adhesion molecules participating in the process of leukocyte-endothelial cell interaction have been targeted for pharmacological interventions for a considerable time. Numerous antibodies have been created to inhibit the function of E-, P-, and L-selectins, ICAM-1, L-integrins, 41- and V3-integrins, and others (reviewed in Ref. 27). So far, none of them has proved beneficial in inflammatory disease, although some clinical trials are still in progress (27).
The Arg-Gly-Asp (RGD) motif is a common recognition sequence for several integrins and has been widely used as a decoy competing with fibronectin, vitronectin, osteopontin, and other proteins containing this sequence (8). Using a modification of atomic force microscopy, which permits quantitative analysis of physical forces between a single monocyte captured on a functionalized cantilever and a single endothelial cell, we measured forces developing in individual ligand-receptor pairs during monocyte-endothelial cell adhesion. We demonstrated that the cyclic (c) RGD inhibits very late antigen (41-integrin; VLA4)/VCAM-1 interaction. In the presence of cRGD and the function blocking monoclonal antibody (mAb) to VCAM-1, we observed a 65% inhibition in work of deadhesion at human umbilical vein endothelial cell (HUVEC) borders and a 52% inhibition at HUVEC bodies, as studied using atomic force microscopy (31). This effect was very similar to that of cRGD alone, suggesting that cRGD did interfere with VLA4/VCAM-1 interaction. In addition, when cRGD and mAbs against P- and E-seletins, ICAM-1, and VCAM-1 were combined, work of deadhesion was further reduced to 75 and 65% in cell borders and bodies, respectively. The data implicated 41-integrin in monocyte-to-endothelial cell adhesion and V3 in TEM. The efficacy of cRGD peptide inhibition of leukocyte adhesion was in the order of IC50 = 100 nM. Transmigration of HL-60 cells was reduced by 60% in the presence of cRGD peptide. Addition of neutralizing anti-V3 or anti-1 antibodies or their combination resulted in a partial but significant inhibition of transmigration, which was not significantly different from the inhibitory effect of cRGD peptide. Both processes were profoundly inhibited by nanomolar concentrations of cRGD peptide. This enticing finding offered a unique opportunity to curtail the development of "fatty streaks" and therefore deserved further investigation into the spectrum of cRGD effects in vitro and in vivo. Based on these in vitro findings, we set to examine in vivo effects of sustained administration of cRGD peptide on monocyte infiltration of the arterial wall in a model of accelerated atherosclerosis, apolipoprotein E (ApoE) knockout mice.
MATERIALS AND METHODS
Animals. Female ApoE/ and their genetic background mice (C57BL/6J) were obtained from Jackson Laboratories. All mice were fed a Western diet throughout the investigation. Wild-type and ApoE/ mice received the following three different types of treatment: cRGD peptide, cRAD peptide (cyclo-Arg-Ala-Asp-D-Phe-Val), or PBS.
RGD treatment in vivo. At the age of 7 wk, we implanted osmotic minipumps (Alzet; Durect, Cupertino, CA) subcutaneously in the dorsum under anesthesia with intraperitoneal injection of ketamine/xylazine. The pumps were either filled with cRGD peptide (20 ApoE/ mice total), cRAD peptide (19 ApoE/ mice total), or PBS buffer (14 ApoE/ mice total). The pumps release 0.25 μl content/h over a period of 14 or 28 days. The fill volume of the pump is 100 μl (or 200 μl). We calculated the concentration of cRGD and cRAD peptide in these pumps in a way that the desired plasma concentration of 100 nmol/l (or 500 nmol/l) is achieved (maintenance dose = target concentration x clearance) using the following formula:
where K0 is the maintenance dose rate or infusion rate; CSS is the target concentration or concentration at steady state; ClP is plasma clearance; Ke is the elimination rate; VD is the volume of distribution; and t1/2 is half-time.
Based on our previous in vitro experiments, the half-maximal working concentration of cRGD peptide is in the 100 nmol/l range; thus, the desired plasma level was chosen as 100500 nM. The half-life of the peptide in the plasma is 1030 min (mainly determined by renal excretion.) Considering the volume and the pump rate of the osmotic pumps, the half-life of the peptide, and the plasma volume of mice, we calculated the concentration of the peptide in the pump to achieve a calculated plasma concentration of 500 nM. In 2-wk experiments, five and four ApoE/ mice received 100 nM cRGD or cRAD peptide, respectively, and seven mice each received 500 nM cRGD or cRAD. In 4-wk experiments, eight mice each received 500 nM peptide treatment.
Biological testing of minipump-delivered cRGD activity. To verify the actual biological activity of the cRGD peptide in the circulation, we measured inhibitory properties of cRGD peptide on platelet aggregation using platelet-rich plasma (PRP) and a Chronolog lumi-aggregometer (Chronolog, Haverton, PA). In brief, whole blood from animals treated with PBS, cRGD, or cRAD was directly drawn using a 26-gauge needle in acid citrate-dextrose (acid citrate-dextrose-blood, 10:1) from the left ventricles of ApoE/ mice before death. The citrated blood was centrifuged at 110 g for 5 min at 21°C. Platelet numbers in PRP, which were not significantly different between animal groups, were adjusted by the addition of platelet-poor plasma, and aggregation was measured at 37°C in the presence of 10 μM ADP.
To verify the stability of cRGD within the minipumps during 1 mo of the functioning, the remnant material from the minipumps was obtained, after animals were killed, and subjected to mass-spectroscopic analysis using a SELDI-TOF technique (Ciphergen). The authentic cRGD peptide was used as a standard.
Histology and histochemistry. The aortas were stained with oil red O solution (oil red O stock solution: 0.5 g oil red O and 100 ml isopropyl alcohol; oil red O working solution: 18 ml oil red O stock and 12 ml distilled water). Residual connective tissue was removed under the microscope. The pins were removed, and the aortas were placed on microscope slides (Fisher Scientific) and covered with a cover glass (Fisher Scientific) using aqueous mounting solution (Biomeda, Foster City, CA).
En face pictures of the aortas were taken with a Nikon microscope and a Nikon digital camera. The atherosclerotic lesions of the small curvature of the aortic arch were analyzed with the Adobe Photoshop 7.0 software or Metamorph software (Universal Imaging). Lesion area was expressed as percentage of stained pixels/surface area.
Kidneys of ApoE/ mice were removed after perfusion-fixation, embedded in paraffin, or snap-frozen. Paraffin or cryosectioning was performed to achieve the thickness of sections of 34 μm. Sections were stained with oil red O. Consecutive glomeruli (50) were evaluated from each animal and graded from 0 to 3+ based on the extent and the staining intensity of oil red O. The total scores were summed up and divided by 50 to obtain an average score per glomerulus.
Immunolabeling of monocytes/macrophages in renal tissue. Samples of renal tissue were fixed in a 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, followed by incubation in 30% sucrose overnight at 4°C. Embedding was performed in an optimum cutting temperature compound (Tissue-Tek, Torrance, CA), and embedded samples were stored frozen at 80°C. Frozen samples were cut into 10-μm-thick sections. Peroxidase activity was blocked by 15 min of incubation with peroxidase block solution (1:10; DakoCytomation, Glostrup, Denmark), and unspecific protein binding was inhibited by 1 h of incubation with PBS-BSA (1% wt/vol). The following antibodies were employed: rat anti-mouse macrophages/monocytes (MCA519, corresponding to an intracellular antigen of mouse macrophages and monocytes, 1:50 in 1% PBS-BSA) for primary incubation and goat anti-rat IgG horseradish peroxidase (STAR72, 1:50 in 1% PBS-BSA) for secondary incubation (Serotec, Oxford, UK). Antibody incubation was performed for 1 h each. Visualization of a positive immunoreaction was made possible by the addition of the standard peroxidase enzyme substrate 3-amino-9-ethylcarbazole (Vector Laboratories, Burlingame, CA), which resulted in a red color reaction product. Hematoxylin solution was used as a nuclear counterstain. PBS washes were performed between each of the above-described steps. Negative controls for all immunolabeling procedures were done by incubation with 1% PBS-BSA instead of the primary antibody. To quantify the monocyte/macrophage infiltration, the number of positive cells per field was evaluated, with the total of 100 glomeruli counted.
In parallel experiments, carotid artery ligation for 3 days was performed in RGD-treated and control ApoE/ mice, and immunohistochemical detection of macrophages as well as oil red O staining of the carotid artery were performed as described above. This model of primary atherosclerosis has been developed by Leidenfrost et al. (16); ApoE-deficient mice fed a Western diet undergo temporary occlusion of the common carotid artery and develop accelerated atherosclerotic lesions containing foam cells, cholesterol clefts, necrotic cores, and fibrous capsules within 23 wk postocclusion.
Statistical analysis. Data are presented as means ± SD. Comparison between groups was performed by ANOVA followed by Tukey's posttest, with P < 0.05 considered as a significant difference.
RESULTS
Bioassay of biological activity of chronically delivered cRGD. First, we verified the biological efficacy of the chronic cRGD treatment by examining whether our cRGD has an inhibitory effect on ADP-induced platelet aggregation by adding cRGD, cRAD, or PBS at different concentrations to whole blood and measuring the ADP-induced coagulation. Plow et al. (22) showed in 1985 that RGD peptides inhibit binding of fibrinogen to ADP-activated platelets. The RGD sequence in the fibrinogen molecule binds to the integrin IIb3. This binding of fibrinogen is competitively inhibited by cyclic pentameric RGD peptides, thereby inhibiting platelet aggregation.
The results are shown in Fig. 1A. These data showed that application of cRGD in vitro blocked aggregation completely at the concentration of 10 μM. Even at the concentration as low as 10 nM there was an 50% inhibition of ADP-induced platelet aggregation. cRAD at a concentration of 10 μM has a measurable but minor effect on aggregation. These findings reconfirmed the inhibitory potency of the cRGD peptide and established this bioassay as a sensitive tool to monitor the plasma levels of cRGD peptide in ApoE/ mice. Figure 1B depicts a family of individual results obtained from the blood of ApoE/ mice chronically treated with cRAD. Chronic infusion of this peptide did not affect ADP-induced aggregation of platelets isolated from animals B11-B16 (Fig. 1). In contrast, 50% of animals in the cRGD-treated group (animals B1B7 in Fig. 1) showed either a complete or a significant inhibition of platelet aggregation (Fig. 1C). This indicates that the delivery of cRGD peptide via osmotic pumps was efficient for at least the most part of the 4-wk period and resulted in a biologically active RGD peptide in the circulation. Surprisingly, in the rest of the animals treated with cRGD peptide, platelet aggregation was not inhibited, although the parameters of fatty streak formation (vide infra) in this group did not differ from those in animals with the inhibited platelet aggregation (data not shown). We have no solid explanation to this finding, except that it may be secondary to the duration of time between blood drawing and testing. It is unlikely, however, that in some animals Alzet minipumps did not function, because all minipumps were examined at the time of death and demonstrated to be near empty. Moreover, mass spectroscopic analysis of the contents of the minipumps showed a single peak of an appropriate molecular mass indistinguishable from that of the authentic peptide (Fig. 2), further confirming the stability of cRGD peptide in the implanted minipumps. In addition, a peak of the mass-to-charge ratio corresponding to that of cRGD peptide was detectable in the serum of animals treated with this peptide but was absent in the serum samples obtained from mice treated with PBS or with RAD peptide (Fig. 2).
Progression of aortic lesions. Wild-type animals fed a Western diet did not develop fatty streaks during 24 wk of the experimental follow-up. ApoE/ mice fed a Western diet for 24 wk exhibited a massive formation of fatty streaks, as judged by the intensity and distribution of oil red O staining of en face aortas. Chronic administration of cRGD peptide at the concentration of 100 for 2 wk showed a trend toward lesser area of atherosclerotic lesion, which did not reach statistical significance; however, the calculated 500 nM for 2 and 4 wk did not appreciably affect the extent of oil red O staining (Fig. 3). Subvalvular plaques were prominent in ApoE knockout mice, and the treatment modality did not appreciably modify the extent of the lesions (data not shown).
Progression of carotid artery lesions. Temporary carotid artery ligation was used to induce formation of atherosclerotic lesions, as shown in Fig 4A, demonstrating a composite image of a carotid artery postligation. ApoE/ mice treated with PBS showed two times the number of macrophages infiltrating the carotid wall than the cRGD-treated animals. Surprisingly, cRAD-treated ApoE/ mice also showed a reduction in the number of infiltrating macrophages (Fig. 4B). This could be attributed to a nonspecific effect of peptides or to a partial antagonistic (RGD-like) action of cRAD peptide.
Progression of nephropathy. ApoE/ mice showed focal accumulation of lipid in glomeruli ranging from segmental to global distribution in the mesangial and/or endocapillary space. Glomerular capillary tufts were focally ballooned out as a result of lipid-laden foam cells. Juxtaglomerular arterioles were focally obliterated with oil red O-positive endothelial cells. oil red O-positive lipid deposits were also seen in occasional cortical and inner medullary interstitial cells. There was a significant decrease in glomerular lipid accumulation in RGD-treated mice (Fig. 5). This treatment also resulted in decreased macrophage accumulation (Fig. 6).
DISCUSSION
The data presented herein partially confirm our previous in vitro findings on the inhibitory action of cRGD peptide on monocyte-endothelial cell interaction. Using a model of accelerated atherosclerosis, ApoE/ mouse, we measured the number of infiltrating macrophages and the degree of lipid accumulation in animals fed a Western diet and receiving a maintenance dose of chronically released cRGD peptide. It was calculated that the osmotic Alzet minipump maintained the calculated concentration of cRGD of 100 and 500 nM. Indeed, the ADP-induced platelet aggregation, used as a bioassay for chemical stability and release of cRGD peptide, showed inhibition of platelet aggregation in 50% of these mice compared with those receiving cRAD. Moreover, mass spectroscopy of the contents of minipumps 1 mo after implantation showed a single peak of the predicted molecular weight corresponding to the authentic cRGD standard. The results showed heterologous activity of cRGD peptide dependent on the site of action. The aortic root displayed resistance to cRGD peptide, whereas the ligation-injured carotid artery and renal microvasculature showed a decrease in macrophage infiltration and lipid accumulation in response to chronic treatment with cRGD peptide.
It has been established that freshly isolated monocytes express not only 2-integrins, as previously believed, but also 1- and 3-integrins (9). Interaction between the 41 (VLA4) and VCAM-1, responsible for the tight monocyte-endothelial cell adhesion, is the main target of cRGD peptide (31). Although this interaction in postcapillary venules is controlled by multiple proatherogenic factors, adhesion of monocytes to the arterial wall is mediated via ANG II (1). This distinction in mediators of monocyte-endothelial cell adhesion in vivo, together with the differences in the physical forces acting on adhesive partners in the arteries and venules, could explain the observed differences in the action of cRGD peptide. Although inefficient at the aortic root and curvature, the peptide was effective in preventing monocytic infiltration of the injured carotid artery. These data are in good agreement with the recently described upregulation of adhesive partners VLA4 and VCAM-1 in the vasculature of ApoE/ mice 37 days after air desiccation injury to carotid arteries and a remarkable efficiency of a neutralizing monoclonal anti-VLA4 antibody, PS/2, in attenuating leukocyte recruitment and neointimal formation (2). The observed selectivity of the RGD effect (significant attenuation of macrophage infiltration of and lipid accumulation in glomeruli and injured carotid artery, with the lesser effect in the aorta) is in good agreement with the predominant distribution of 41-integrin (VLA4) to the renal vasculature and injured vessels (2, 7). In addition to this distinction, we clearly appreciate that there are multiple other receptors participating in the monocyte-endothelial cell adhesion and transmigration, and their contribution to the process may be insensitive to cRGD peptide, thus reducing its efficacy. Yet, at the vascular sites enriched in VLA4, the cRGD-dependent mechanism of adhesion and transmigration may be dominant. Under these circumstances, the active concentration of cRGD peptide is in the high nanomolar range. Increasing the concentration of cRGD peptide continuously and delivering it chronically could, however, result in an untoward consequence because of the potential endothelial cell detachment.
The vulnerability of renal microvasculature with the rapid accumulation of lipids has recently been demonstrated. It was shown that oxidized low density lipoprotein (LDL) stimulates monocyte adhesion to glomerular endothelial cells (11). In this vein, nitrosylation of LDL cholesterol by peroxynitrite takes place, especially in atherosclerotic plaques, contributing to lipid peroxidation (14). Because one of the prominent receptors for oxidized LDL on endothelial cells, LOX-1, is upregulated by ANG II or shear stress, activation of the renin-angiotensin system in many forms of hypertension may be causatively linked to the accelerated atherogenesis (12). In fact, recent findings by Bouriquet et al. (3) demonstrate that, in the absence of hyperlipidemia, inhibition of eNOS alone is sufficient to induce the deposition of sudan black-positive lipid droplets in arcuate and interlobular renal arteries, but not in afferent arterioles, resulting in increased vascular wall thickness. This study importantly demonstrated the role of NO generation in atherosclerotic damage to the medium-size renal arteries. In the case of ApoE/ mice, which exhibit systemic endothelial cell dysfunction and are thus predisposed to monocyte adhesion and egress, their renal vascular bed appears to be effectively protected from macrophage infiltration by the cRGD peptide. This protection also resulted in the reduced accumulation of lipids in the glomeruli, the precursor of lipid nephropathy.
One of the unexpected findings of this study was related to the effect of cRAD partially mimicking that of cRGD and raising the question of specificity of the cRGD effect. Rather than attributing it to a nonspecific action of the peptides, we believe that several possible scenarios explaining this effect of cRAD peptide may exist. First, the critical role of aspartic acid in the action of RGD peptides has been unequivocally demonstrated (25), and yet this amino acid is present in the "control" peptide. Although Samanen et al. (24) showed that RAD peptide is less potent than RGD peptide with respect to platelet aggregation, the peptide containing the sequence YMERSRAD within the IIb3 binds fibrinogen and blocks fibrinogen binding to platelets (19). At the same time, atherosclerotic lesions were found to be reduced in fibrinogen-deficient mice raised on the ApoE/ background (17). This latter finding is controversial, however, because fibrinogen deficiency did not affect the rate of atherosclerosis in ApoE/ mice, according to other investigators (29). These data collectively question cRAD peptide as an appropriate negative control for cRGD peptide and highlight their partial agonism.
Our findings extend the previous usage of RGD peptides in blocking cell-to-matrix adhesion, inhibiting angiogenesis and platelet aggregation, and as probes for imaging of vascular injury (4, 6, 10, 21, 23) and raise the possibility of their therapeutic applicability in suppressing monocytic infiltration of the vascular wall, the prerequisite for the formation of atherosclerotic plaque, especially in the vessels with intimal injury and in renal microcirculation. Therefore, our observation on the potency of cRGD peptide in inhibiting monocyte-endothelial cell interaction makes this nontoxic and nonimmunogenic compound an attractive alternative to the neutralizing antibodies. Its added bonus is inhibition of IIb3 and platelet aggregation, an important target in patients with atherosclerosis.
GRANTS
These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45695 (M. S. Goligorsky), DK-52783 (M. S. Goligorsky), and DK-064863 (S. V. Brodsky), the Jaeckstaedt Foundation (D. Patschan), American Heart Association Grant 0256161T (K. M. Lerea), and the Westchester Artificial Kidney Foundation.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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