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Amyloid precursor protein (APP) is a ubiquitously expressed type one integral membrane protein. It has the ability to bind numerous extracellular matrix components and propagate signaling responses via its cytoplasmic phosphotyrosine, 682YENPTY687, binding motif. We recently demonstrated increased protein levels of APP, phosphorylated APP (Tyr682), and beta-amyloid (Aβ) in brain vasculature of atherosclerotic and Alzheimer’s disease (AD) tissue co-localizing primarily within the endothelial layer. This study demonstrates similar APP changes in peripheral vasculature from human and mouse apoE−/− aorta suggesting APP-related changes are not restricted to brain vasculature. Therefore, primary mouse aortic endothelial cells (PAEC) and human umbilical vein endothelial cells (HUVEC) were used as a model system to examine the function of APP in endothelial cells. APP multimerization with an anti-N-terminal APP antibody, 22C11, to simulate ligand binding stimulated a Src kinase family dependent increase in protein phosphotyrosine levels, APP phosphorylation, and Aβ secretion. Furthermore, APP multimerization stimulated increased protein levels of the proinflammatory proteins, cyclooxygenase (COX)-2 and vascular cell adhesion molecule (VCAM)-1 also in a Src kinase family dependent fashion. Endothelial APP was also involved in mediating monocytic cell adhesion. Collectively, these data demonstrate that endothelial APP regulates immune cell adhesion and stimulates a tyrosine kinase-dependent response driving acquisition of a reactive endothelial phenotype. These APP-mediated events may serve as therapeutic targets for intervention in progressive vascular changes common to cerebrovascular disease and AD.
Endothelial cells play an important role in both the maintenance and inflammatory responses of the peripheral and brain vasculature. They are integral in clot formation, angiogenesis, and controlling blood pressure by mediating vasodilation or constriction. Importantly, endothelial cells are also the site for immune cell adhesion and eventual infiltration into the tissue.
Adhesion based activation is phenotypically important in numerous cell types, but particularly for immune cells and endothelial cells. Previously, we demonstrated that monocytic cells use amyloid precursor protein (APP) to mediate acquisition of an adhesion based proinflammatory phenotype (Sondag and Combs, 2004). Endothelial cells also express APP and surface localization increases following stimulation with proinflammatory cytokines, such as interleukin (IL)-1β (Goldgaber et al., 1989; Forloni et al., 1992). APP overexpression in endothelial cells is reportedly toxic (Jahroudi et al., 1998) and these cells express the secretase enzymes required to generate beta amyloid (Aβ) peptides (Davies et al., 1998). Moreover, we recently demonstrated increased immunoreactivity of APP, pAPP (APP phosphorylated at tyrosine residue 682) and Aβ within the cerebrovasculature, particularly in endothelial cells, of both atherosclerotic and AD tissue (Austin and Combs, 2008). More importantly, adhesion of THP-1 monocytic cells was partially dependent upon endothelial APP expression (Austin and Combs, 2008). These data suggest that APP regulates not only cell-cell adhesion but also modulates the proinflammatory phenotype of endothelial cells.
APP is a highly conserved and ubiquitously expressed type 1 integral membrane protein that has been suggested to function in cellular adhesion. It has the ability to interact with numerous adaptor proteins (Borg et al., 1996; Howell et al., 1999; Russo et al., 2002; Scheinfeld et al., 2002; Venezia et al., 2004) and bind several components of the extracellular matrix (Kibbey et al., 1993; Williamson et al., 1995; Beher et al., 1996). Furthermore APP contains a conserved 682YENPTY687 cytoplasmic motif similar to nonreceptor tyrosine kinases which may be involved in propagating signaling responses.
Taken together, these data and our prior work demonstrating the role of APP as an adhesion receptor in immune cells supports the hypothesis that endothelial APP is involved in mediating immune cell adhesion and subsequent acquisition of a reactive phenotype which may occur during progressive vascular dysfunction seen in cardiovascular/cerebrovascular disease and AD. To further examine the role of APP within endothelial cells we have now utilized primary murine aortic endothelial cells (PAEC) and human umbilical vein endothelial cells (HUVEC) as two common endothelial cell in vitro model systems. Multimerization of endothelial APP stimulated increased expression and secretion of proinflammatory proteins. Further, adhesion of monocytic cells to a HUVEC monolayer was partially dependent on endothelial APP. Understanding the role of endothelial APP in regulating cell-cell adhesion and subsequent changes in endothelial phenotype may provide a therapeutic target for diseases which involve vascular dysfunction and immune cell infiltration.
The anti-APP (22C11) antibody, anti-von Willebrand factor and the mouse IgG1 isotype control were purchased from Chemicon (Temecula, CA). The phospho-tyrosine (pTyr) antibody was purchased from Upstate (Lake Placid, NY). The anti-Aβ, anti-cyclooxygenase (COX)-2, anti-cSrc, anti-vascular cell adhesion molecule (VCAM)-1 antibodies and the horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The anti-smooth muscle actin antibody was purchased from Novus Biologicals, Inc. (Littleton, CO). Anti-APP antibody was purchased from Zymed Laboratories (San Francisco, CA). The anti-pSrc antibody was from Cell Signaling Technologies, Inc. (Danvers, MA) and the anti-smooth muscle actin antibody from Novus Biologicals, Inc. (Littleton, CO). The anti-α-tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pAPP was generated by immunizing rabbits against the phospho-682 phosphoYENPTY687 sequence of human APP695. Affinity purified anti-pTyr682APP antibodies were used. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyrimidine (PP2) and the anti-inducible nitric oxide synthase (iNOS) antibody were purchased from Alexis Biochemicals (San Diego, CA).
Apptm1Dbo/J homozygous (APP−/−), Apoetm1Unc/J homozygous (apoE−/−) and wild type mice (C57BL6/J) were purchased from Jackson Laboratory. Mice were provided food and water ad libitum and housed in a 12 hour light/dark cycle. Mice were sacrificed and abdominal aortas were collected at 8 months, immersion fixed for 24 hours in 4% paraformaldehyde, cryoprotected through 2 successive 30% sucrose changes, and serially sectioned (40μm) via freezing microtome. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996)
Two individual human abdominal aorta samples were obtained from the University of North Dakota, Dept. of Pathology Tissue Bank and fixed and sectioned as described above. The investigation conforms with the principles outlined in the Declaration of Helsinki. All animal use and human tissue use was approved by the University of North Dakota IACUC, IBC/IRB, respectively.
THP-1 monocytic cells, commercially available from the American Type Culture Collection (ATCC, Manassas, VA), are derived from the peripheral blood of a human with acute monocytic leukemia. THP-1 cells were grown as previously described (Austin and Combs, 2008). HUVEC were obtained from Sciencell Research Laboratories (Carlsbad, CA). HUVEC were grown in endothelial cell media (ECM) which was made from RPMI-1640 media supplemented with 10% FBS, 1% endothelial cell growth supplement (ECGS, Sciencell Research Laboratories, Carlsbad, CA) and 1.5 μg/ml penicillin/streptomycin/neomycin. PAEC were obtained as previously described by McGuire and Orkin (McGuire and Orkin, 1987). Briefly, the abdominal aorta was removed from 8 month C57BL/6 (wild-type) or APP−/− mice and rinsed with HBSS. Periadventitial fat was removed and rings less than 2mm were cut. These aortic explants were placed onto a matrigel (BD Biosciences, Bedford, MA) substrate in ECM for 4–7 days before explants were removed. Endothelial cells were allowed to grow for several days before collection from the matrigel using dispase (BD Biosciences, Bedford, MA). After collection PAEC were then cultured on poly-L-lysine coated tissue culture plastic or coverslips for use.
To increase the amount of surface APP on the THP-1 monocytic cells, HUVEC, and PAEC were stimulated with 25 ng/ml LPS for 2 hours and 25 ng/ml TNF-α for 3–4 hours, respectively, at 37°C prior to use in experiments.
In order to fluorescently label THP-1 cells for use in the adhesion assays, previously stimulated THP-1 cells were incubated with DNA labeling dye from the Cyquant proliferation assay (Molecular Probes, Eugene, OR) for 10 min at 37°C prior to use.
Endothelial cells were collected using 0.25% EDTA/Trypsin and plated overnight allowing them to attach and equilibrate before use in experiments. Cross-linking of endothelial surface APP was achieved by incubating HUVEC and PAEC with or without an N-terminal anti-APP antibody (22C11, 1μg/mL) or mouse IgG1 (1μg/mL) isotype control for 30 min or 24 hours at 37°C.
PP2, a non-competitive inhibitor of ATP, is a selective Src family kinase inhibitor which binds adjacent to the ATP binding site (Hanke et al., 1996; Zhu et al., 1999; Karni et al., 2003). To inhibit Src kinase activity, endothelial cells were pretreated with PP2 30 minutes prior to and concomitantly during duration of experiments.
To perform immunohistochemistry, sections (40μm) were immunostained using anti-von Willebrand factor, anti-smooth muscle actin, anti-APP and anti-pAPP and visualized using either Vector VIP, DAB, or Vector Blue as the chromagens (Vector Laboratories, Burlingame, CA). Secondary antibody only staining was performed as non-specific staining controls.
To perform immunocytochemistry, cells were fixed with 4% paraformaldehyde (30 min, 37°C) and immunostained using anti-pAPP, anti-Aβ, anti-VCAM-1, anti-COX 2, anti-von Willebrand factor or anti-smooth muscle actin antibodies as previously described (Austin and Combs, 2008). 4′, 6′-diamidino-2-phenylindole (DAPI) was used to visualize the nucleus. Images were captured using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Thornwood, NY).
Cells were stimulated as described above. Western blot analysis was performed as previously described (Austin and Combs, 2008). Optical density of protein bands from 5–6 independent experiments were normalized against a protein loading control (α-tubulin) using Adobe Photoshop software and statistical analysis was performed. Data are presented as mean values (±SD), and statistical significance was determined by unpaired t test.
Cells were stimulated as described above. Immunoprecipitations were done as previously described by Austin and Combs (Austin and Combs, 2008). Immunoprecipitates were then used for Western analysis as described above.
Media was collected from HUVEC following 24 hour stimulation. Levels of human interleukin (IL)-1β and Aβ1-40 and Aβ1-42 were detected using commercially available ELISA kits from eBioscience (San Diego, CA) per manufacturer’s instructions. Statistical analysis of data was performed using an unpaired ANOVA with Tukey-Kramer’s post hoc comparison. Data are represented as mean (±SD) (*p<0.001)
In order to assess effects of APP cross-linking on cellular proliferation we utilized Cyquant NF cell proliferation assay (Molecular Probes, Eugene, OR). Cells were stimulated overnight and the proliferation assay performed according to manufacturer’s instructions. Statistical analysis of data was performed using an unpaired ANOVA with Tukey-Kramer’s post hoc comparison. Data are represented as mean (±SD).
To determine cell viability following 24 hour cross-linking stimulation, the cellular release of lactate dehydrogenase (LDH) was measured using a commercially available nonradioactive assay (Promega, Madison, WI). Absorbance measurements were taken at 490nm. Statistical analysis of data was performed using an unpaired ANOVA with Tukey-Kramer’s post hoc comparison. Data are represented as mean (±SD).
To assess tissue adhesion a modified Stamper-Woodruff adhesion assay was used (Stamper and Woodruff, 1976). Briefly, serial aortic sections from apoE−/−, APP +/−, APP−/− and APP+/+ (wild-type/control) aortas (40μm) were mounted onto subbed slides, 3 aortas per background. Sections were incubated with/without 22C11 (anti-APP) or mouse IgG1 (isotype control) antibodies in phosphate buffered saline (PBS) solution (PBS containing 0.5% BSA, 2% FBS) for 1 hour, room temperature to bind surface APP. Sections were rinsed with PBS solution before an hour-long incubation with stimulated and labeled THP-1 cells (as described above) at room temperature with gentle agitation (50 rpm). Sections were rinsed three times with PBS solution and adherent cells fixed with 4% paraformaldehyde, 20 minutes, 37°C. Images were captured using a Zeiss (Zeiss, Thornwood, NY) LSM 510 Meta confocal microscope.
Number of adherent cells was determined in a double blinded fashion by counting the number of cells adherent to vessel walls of serial sections in several fields of five independent sections from three animals from each background. Data are presented as mean values (±SD), and statistical significance was determined by unpaired ANOVA with Tukey-Kramer’s post hoc comparison (* p <0.001).
To assess cell-cell adhesion, a monolayer of HUVEC were plated in 96 well plates then incubated with or without the N-terminal anti-APP antibody or IgG1 isotype control to bind APP and/or downregulate cell surface APP for 1 hour at 37°C. HUVEC were then incubated with a cell suspension of labeled THP-1 cells (as described above) for one hour at 37°C, followed by three rinses with ECM. Adhesion was quantitated by measuring fluorescence of adherent (fluorescently labeled) cells at 490 nm. Statistical analysis of data was performed using an unpaired ANOVA with Tukey-Kramer’s post hoc comparison. Data are represented as mean (±SD) (*p<0.05).
To begin addressing a broader role of APP in the peripheral as well as brain vasculature, levels of APP in human atherosclerotic aorta were examined by immunostaining with anti-APP and tyrosine 682 phosphorylated (pAPP) antibodies. The aortas revealed strong immunoreactivity for both APP and pAPP (Figure 1A). Indeed, using von Willebrand factor and smooth muscle actin immunoreactivity to visualize the layers of the aorta we verified that the majority of the immunoreactivity was confined to the intimal layer (Figure 1B). Interestingly, although subendothelial deposition of von Willebrand factor has been reported prior, APP/pAPP immunoreactivity also extended beyond the immediate single cell layer of the endothelium into the intima (Nizheradze, 2006). These data confirmed that changes in APP phosphorylation and immunoreactivity observed in brain vasculature in prior work can be extended to peripheral vasculature implying a broader role for APP in endothelial biology (Austin and Combs, 2008).
In order to study a more controlled model of vascular atherosclerotic changes, apoE−/− mice were next examined for changes in APP. Abdominal aortas were collected from apoE−/− and age-matched wild-type mice at 8 months of age. Prior data by Reddick et al demonstrated that apoE−/− mice maintained on a standard chow diet develop increasing plaque number and complexity with age, with plaques present in the carotid arteries, abdominal aorta and iliac arteries in animals at 9 months (Reddick et al., 1994). Therefore, abdominal aortas from 8 month apoE−/− mice were assessed to determine whether changes in APP were prior to robust plaque deposition. Similar to the observations from the human samples, apoE−/− aorta demonstrated strong APP immunoreactivity compared to wild type controls (Figure 2). Again, this reconfirms prior finding from both human and rodent brain vasculature (Austin and Combs, 2008). Interestingly, APP immunoreactivity in the apoE−/− aorta was increased in both the intimal and adventitial layers compared to controls. The adventitial change was not entirely unexpected given prior reports of increased APP expression in both fibroblasts and immune cells during stressful conditions (Johnston et al., 1994; Vehmas et al., 2004). However, only the intimal layer of the apoE−/− vasculature displayed increased pAPP immunoreactivity which colocalized with the endothelial marker, von Willebrand factor (Figure 2). Intimal specific pAPP immunoreactivity suggests that APP serves unique functions within the cells of different aortic layers. To quantify the differences demonstrated by immunohistochemistry, Western blot analyses were performed. As predicted, apoE−/− mouse aortic tissue demonstrated significantly higher levels of both APP and pAPP protein compared to wild type controls (Figure 3).
Although the histological and Western data supported the hypothesis that APP functions to contribute to vascular dysfunction, these observations remained correlative. To begin directly defining the role of APP in endothelial cell activation and increased adhesiveness, a modified Stamper-Woodruff adhesion assay was used to examine adhesion of human monocytic lineage THP-1 cells to aortic endothelium from apoE−/−, APP+/+ (C57BL/6J wild-type animals), APP+/−, and APP−/− mice (Figure 4). Based upon the high degree of homology shared between the amino acid sequence of mouse and human APP we reasoned that murine endothelial APP would interact with human monocytic proteins (Yamada et al., 1987). Significantly more monocytic cells adhered to apoE−/− tissue compared to the APP+/+ wild-type controls and this was attenuated by pre-incubating the aortic sections from apoE−/− and APP+/+ mice with an anti-N-terminal APP antibody, 22C11, to bind surface APP (Figure 4). The role of APP was reconfirmed using tissue from APP genetically depleted animals. Monocytes demonstrated a gene dosage-dependent decrease in the number of adherent cells to aortic endothelium from APP+/+ (C57BL/6J wild-type), APP+/− and APP−/− mice (Figure 4). Importantly, similar findings of APP-dependent adhesion have been reported from AD brain vasculature as well as cerebrovasculature from atherosclerotic apoE−/− animals verifying again a broad role for APP in vascular biology (Austin and Combs, 2008).
Based upon the fact that endothelial expression of APP increases following proinflammatory stimulation (Goldgaber et al., 1989; Forloni et al., 1992), the current histological findings, and our prior results of increased endothelial expression of APP, pAPP and Aβ in the cerebrovasculature of both atherosclerotic and AD tissue (Austin and Combs, 2008) we hypothesized that endothelial APP mediates an activating response in these cells. To begin studying the function of endothelial APP, PAEC and HUVEC were selected as well-characterized in vitro model systems for assessing endothelial biology. Since minimal APP is localized to the plasma membrane basally, PAEC and HUVEC were pretreated with TNFα for 3 hours before any stimulations were performed. Non-permeablizing immunocytochemistry verified that TNFα stimulation increased the localization of APP to the surface of endothelial cells (Figure 5A, B). Further, immunocytochemistry using antibodies recognizing von Willebrand factor, an endothelial marker, or smooth muscle actin, a smooth muscle marker, verified that the cultures were routinely at approximately 98% purity (Figure 5C).
To define the role of APP in the absence of a well-defined stimulatory ligand we elected to simulate ligand binding through the use of the N-terminal anti-APP antibody, 22C11, which would induce APP multimerization and subsequently activate a signaling response. This approach has been successfully used in the past to stimulate APP-mediated signaling and proinflammatory changes in monocytic cells (Sondag and Combs, 2004). PAEC and HUVEC were stimulated with or without 22C11 or an IgG1 isotype control for 30 minutes, to assess changes in phosphorylation and processing of APP. Multimerization stimulated increased immunoreactivity for tyrosine phosphorylated APP as well as the proteolytic fragment Aβ (Figure 6). Since prior work using brain tissue from apoE−/− and AD patients demonstrated increased association of APP with the tyrosine kinase, Src, (Austin and Combs, 2008) a similar signaling response was examined using the cell lines. Pretreatment of the PAEC or HUVEC with the Src family member inhibitor, PP2, attenuated the multimerization induced change in immunoreactivity for APP phosphorylation and Aβ demonstrating that these were Src kinase dependent (Figure 6).
To quantify the immunocytochemical results of multimerization induced changes in APP phosphorylation, Western analyses of HUVEC was performed following stimulation with or without 22C11 or IgG1 isotype control. APP multimerization stimulated statistically increased levels of tyrosine phosphorylated proteins, specifically increased phosphorylation of both APP and the active, phosphorylated form of Src in a Src kinase dependent manner (Figure 7). These results suggested that APP multimerization lead to an association between endothelial APP and the tyrosine kinase, Src, allowing for subsequent Src-mediated phosphorylation of APP. To verify an interaction of APP with Src, immunoprecipitations of APP following 22C11 or IgG1 isotype control stimulations were next performed. These pull down assays indeed demonstrated an increased association of APP with Src following 22C11-induced multimerization of APP (Figure 8). The multimerization dependent increase in APP-Src association, Src activation (as assessed by phosphorylation) and Src-dependent APP phosphorylation in the endothelial cells strongly suggested that APP mediated a tyrosine kinase dependent signaling response required for endothelial activation.
To address whether the APP stimulated signaling response lead to an altered endothelial phenotype, changes in levels of common reactive endothelial marker proteins were examined after multimerization-induced activation. Immunocytochemistry and Western blot analyses were performed on PAEC and HUVEC following stimulation with or without 22C11 or IgG1 isotype control for 24 hours. APP multimerization stimulated increased immunoreactivity for both COX-2 and VCAM-1. Immunoreactivity for both COX-2 and VCAM-1 were markedly increased in the cells treated with 22C11 compared to the IgG1 isotype control (Figure 9). To determine if these multimerization-induced proinflammatory changes were dependent upon Src kinase activity, cells were pretreated with PP2 before stimulation. Src kinase inhibition significantly attenuated the multimerization induced changes in immunoreactivity for COX-2 and VCAM-1 (Figure 9).
To quantify the immunocytochemical changes, Western analyses of both PAEC and HUVEC following stimulation with or without 22C11 or IgG1 in the absence or presence of PP2 pretreatment were then performed. Western analyses confirmed that 22C11 induced multimerization stimulated a significant Src kinase-dependent increase in COX-2 and VCAM-1 protein levels but not inducible nitric oxide synthase (iNOS) compared to IgG1 isotype controls (Figure 10). To further confirm the role of APP and the specificity of the 22C11 antibody, Western blot analyses were performed from wild type (APP+/+) C57BL/6J and APP−/− PAEC following stimulation with or without 22C11 or IgG1. Importantly, multimerization using the 22C11, N-terminal anti-APP antibody had no ability to increase protein levels in APP−/− cells (Figure 11a). Conversely, APP stimulation significantly increased protein levels of COX-2 and VCAM-1 but not iNOS in wild type cells (Figure 11b–d). These data verified not only the specificity of the 22C11 antibody as an APP stimulatory ligand but also the necessity of APP-mediated signaling for the change in specific proinflammatory protein levels.
Another important aspect of a reactive endothelial phenotype is their secretion of proinflammatory mediators such as chemokines and cytokines into the extracellular environment. These secreted molecules can act in an autocrine and paracrine fashion, further stimulating the endothelial cells and neighboring cells. Therefore, the ability of APP multimerization to stimulate acquisition of a reactive secretory phenotype was next determined. In particular, secreted levels of interleukin 1-β (IL-1β), a well-characterized proinflammatory cytokine and activator of endothelial cells and the APP proteolytic fragments, Aβ1-40 and Aβ1-42, were quantified. Aβ has been shown to have numerous effects on endothelial cells, including: activation, increased adhesive properties as well as death (Thomas et al., 1996; Farkas et al., 2003; Folin et al., 2005; Gonzalez-Velasquez and Moss, 2008). ELISA was used to quantify media levels of secreted IL-1β, Aβ1-40, and Aβ1-42 levels from HUVEC following stimulation with or without 22C11 or IgG1 isotype control. APP multimerization stimulated a Src dependent increase in secretion of IL-1β as well as Aβ1-40 (Figure 12) while levels of Aβ1-42 were undetectable (data not shown). These data confirmed that the APP stimulated, tyrosine kinase mediated signaling response was responsible for a plethora of changes in proinflammatory protein expression and secretion in endothelial cells.
As mentioned earlier, prior reports have suggested a role for APP in regulating adhesion based upon its ability to bind components of the extracellular matrix (Kibbey et al., 1993; Williamson et al., 1995; Beher et al., 1996; Sondag and Combs, 2004). To broaden the understanding of endothelial APP, a more functional assessment of APP was next examined. Specifically, the role of APP in mediating immune cell-endothelial cell adhesion, endothelial proliferation, and endothelial toxicity were all examined.
Since the modified Stamper Woodruff assay (Figure 4) had already demonstrated a requirement of APP in regulating THP-1 monocyte-endothelial adhesion in fixed aortic tissue, this cell line was used again. To remove the caveat of fixed tissue, live HUVEC cells were employed to assess monocyte-endothelial interaction. The N-terminal anti-APP 22C11 antibody was used to bind endothelial HUVEC APP effectively competing for subsequent interaction with the THP-1 cells. As expected, there was a significant decrease in the number of adherent monocytic cells to the HUVEC monolayer that had been preincubated with 22C11, the anti-APP antibody (Figure 13). Interestingly, APP multimerization did not significantly affect proliferation or toxicity demonstrating that the protein has a limited, specific function for altering the phenotype of endothelial cells (Figure 13). This data verified that endothelial APP was involved in regulating immune cell-endothelial interaction and that the subsequent, stimulated signaling response mediated by APP lead to increased proinflammatory protein expression and secretion but not cell loss or proliferation.
APP is a ubiquitously expressed and highly conserved protein and has been suggested to function in cell-cell or cell-matrix adhesion. We recently reported that cerebrovasculature of AD and atherosclerotic tissue demonstrated disease related changes in APP expression and phosphorylation within the endothelial layer (Austin and Combs, 2008). Furthermore, adhesion of monocytic cells to brain endothelium was partially APP-dependent (Austin and Combs, 2008). This study extends the brain findings to include peripheral vasculature demonstrating a common role for APP-mediated adhesion and tyrosine kinase-based endothelial activation may occur in vasculature in general. Using two different common in vitro endothelial systems, HUVEC and PAEC, as general models of endothelial cell behavior, we demonstrated that multimerization of APP, to simulate ligand binding, stimulated endothelial cells to acquire a Src-dependent reactive phenotype characterized by a robust increase in phosphorylation and processing of APP, increased protein levels of proinflammatory mediators such as COX-2 and VCAM-1, and increased secretion of IL-1β and Aβ1-40. Moreover, adhesion of monocytic cells to aortic endothelium or a HUVEC monolayer was partially APP dependent. Taken together these data suggest that endothelial APP mediates adhesion as well as acts as a proinflammatory receptor in these cells.
Although an N-terminal anti-APP antibody was used to simulate ligand binding, the physiologic relevance of the characterized endothelial biology will be enhanced through ultimate identification of a physiologic APP ligand. Although characterizing an APP ligand, if any, is beyond the scope of the current study, it is interesting to note that APP has been show to interact with itself as well as its cleavage products. Several groups have demonstrated an interaction of fibrillar and soluble Aβ with APP which leads to APP clustering and a stimulated signaling response (Chung et al., 1999; Lorenzo et al., 2000; Wagner et al., 2000; Van Nostrand et al., 2002; Lu et al., 2003; Gralle et al., 2009). It is possible that such an event could occur in endothelial cells in vivo. Certainly, the endothelial results in the current study demonstrated that Aβ1-40 secretion increased following APP multimerization. Possibly, this increased Aβ could act in an autocrine or paracrine feed-forward fashion to further stimulate the endothelial cells or neighboring cells.
Interestingly, numerous groups have demonstrated that membrane-bound APP can form homo- and heterodimers with APP and other APP family members in a cis (same cell) or trans (opposing cell) fashion (Rossjohn et al., 1999; Scheuermann et al., 2001; Lu et al., 2003; Wang and Ha, 2004; Soba et al., 2005). Furthermore, Soba et al demonstrated APP homo and hetero-dimerization in a trans fashion lead to increased adhesion of fibroblasts (Soba et al., 2005). These data suggest that an interaction between APP on both monocytes and endothelial cells could be involved in mediating transdimerization adhesion and responsible for stimulating the tyrosine kinase-based activation response in endothelial cells. Alternatively, our prior work demonstrated that APP is recruited into a multimeric complex with β1-integrin upon adhesion based stimulation of monocytes with a collagen I substrate (Sondag and Combs, 2004). This data suggests that APP may be involved in adhesion by recruitment into a complex with a more conventional adhesion receptor. Further characterization of the exact monocyte-endothelial protein-protein interaction that explains the dependence on APP expression for adhesion is needed to fully define the role of APP in cell-cell adhesion.
The ability of APP to stimulate acquisition of a reactive phenotype in endothelial cells has relevance to multiple disease conditions. For example, we have demonstrated disease related changes in endothelial APP expression, phosphorylation and processing in both brain (Austin and Combs, 2008) and peripheral vasculature in AD and cerebrovascular/cardiovascular disease tissue from humans and apoE−/− mice. Both cerebrovascular/cardiovascular disease and AD involve inflammation and vascular dysfunction. Immunoreactivity was not restricted to the endothelial layer alone, however, and increases were observed further into the intimal layer. One possibility is that the proinflammatory environment in the vessels stimulated other cell types, including smooth muscle cells, to increase APP expression. Indeed, stimulation leading to increased expression of APP has been reported (Goldgaber et al., 1989; Shoji et al., 1990; Banati et al., 1993; Banati et al., 1994; Sondag and Combs, 2004). The small sample size of tissue allowed only a qualitative assessment of human APP changes but the larger sample number of apoE−/− aortas permitted Western quantification to verify a significantly greater amount of murine APP and pAPP levels in the apoE−/− animals compared to controls. Although the current study and our prior work (Austin and Combs, 2008) have only examined abdominal aorta and brain vasculature from AD versus control tissue and apoE−/− versus wild type animals it is intriguing to speculate that the APP related changes observed are not unique to any particular localization of vasculature and rather represent a common mechanism for vascular inflammation in diseases such as cardiovascular disease and AD. Indeed, it was with this general vascular role in mind that we elected to use primary mouse aortic endothelial cells and human umbilical vein endothelial cells as our model systems. We are aware that brain vascular endothelial cells may indeed differ with respect to APP function compared to either of these two cells. However, based upon our prior and current results of similar endothelial APP upregulation in both brain and peripheral vasculature in AD brains and atherosclerotic tissue it appears these changes are common to several endothelial cell types. At the very least, these data provide a baseline for general APP function in endothelial cells and further dissection of individual endothelial differences can now be explored.
It is important to reiterate that the study sought to understand the biology of APP as an endothelial protein. We are aware, however, of the many findings demonstrating APP proteolytic fragments have direct ligand-type effects on endothelial cells resulting in dysfunction relevant to AD and cerebrovascular disease and have not attempted to readdress that biology in this study (Thomas et al., 1996; Iadecola et al., 1999; Price et al., 2001; Elesber et al., 2006). Rather, this data suggests that a parallel effect of endothelial APP function may be occurring in addition to any of those introduced by APP fragment or Aβ stimulation to coordinately regulate endothelial and vascular function. For example, it is interesting to speculate that one such vascular effect that might also be attributed to changes in endothelial APP may be contribution of endothelial secreted Aβ to vascular amyloid depositon. Cerebral amyloid angiopathy (CAA) and capillary CAA (CapCAA) are well characterized occurrences in AD brains. CAA and CapCAA involve the accumulation of predominantly Aβ1-40 in leptomeningeal and cortical arteries and capillaries, respectively (Yamaguchi et al., 1992; Attems and Jellinger, 2004; Attems et al., 2004). Therefore, it is possible that Aβ 1-40 secreted by endothelial cells is one source for the vascular amyloidosis that occurs during disease in addition to peptide contributed from other cells in the brain or the vasculature.
Another intriguing possibility of consequences of increased endothelial APP may be enhanced immune cell adhesion and diapedesis. The adhesion results certainly support this notion. Based upon our data, it is feasible that changes in endothelial cell surface APP in response to inflammatory changes facilitates increased immune cell attachment and diapedesis into tissue. Moreover, APP may represent one of many endothelial cell surface receptors that regulates adhesion and migration of immune cells from the blood into a target tissue. This suggests that targeting APP mediated expression and/or signaling in the endothelial layer will provide therapeutic targets for early treatment in diseases involving vascular inflammation and/or immune cell infiltration.
Determining the timeline and histological localization of APP changes as related to vascular inflammation, atherosclerotic plaque deposition, or cerebral amyloid angiopathy will be necessary to better determine the extent of involvement of APP on the progression of vascular dysfunction as an adhesion mediator and as an endothelial proinflammatory receptor. Further functional studies can then be performed by cell-type specific manipulation of APP expression or its stimulated signaling response to help define the precise role of endothelial APP in disease-associated vascular changes. This study expands upon prior reports of tyrosine phosphorylated, Src-associated APP within the cerebrovasculature of human AD brains and atherosclerotic apoE−/− mouse brains to include similar findings from peripheral aorta from humans and apoE−/− mice and two different endothelial in vitro model systems. Collectively, these data offer continued support for the hypothesis that APP contributes to endothelial phenotype changes that occur during progression of vascular dysfunction in both brain and peripheral vessels in cardiovascular/cerebrovascular disease and AD.
This work was supported by the National Institutes of Health [2P20RR017600, 1R01AG026330]; and the North Dakota National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR) [RRNI EPS-0447679].
S.A. Austin, Department of Pharmacology, Physiology & Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202.
M.A. Sens, Department of Pathology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202.
C.K. Combs, Department of Pharmacology, Physiology & Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202.