Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes

Abstract

Objective:

Reelin and its receptor Apoer2 play a prominent role in endothelial cell dysfunction by promoting leukocyte–endothelial cell adhesion, an important component of the inflammatory process underlying atherosclerosis. We therefore hypothesized that pharmacological depletion of circulating Reelin represents a novel therapeutic strategy to impede the progression of atherosclerosis.

Approach and Results:

In vitro studies demonstrated that human plasma induced monocyte adhesion to endothelial cells, while Reelin-depleted plasma had no effect on monocyte adhesion. Signaling analysis revealed that Reelin activated Dab2, PI3K, Akt and NF-κB cascade to promote the expression of adhesion markers (E-selectin, ICAM-1, and VCAM-1). Intravital microscopy confirmed decreased leukocyte-endothelial adhesion in mice treated with Reelin antisense oligonucleotide (ASO). In vascular smooth muscle cells, Reelin induced Stat3 phosphorylation to promote cell proliferation, which is another hallmark of atherosclerotic plaque progression. To investigate if Reelin pharmaceutical depletion protects against atherosclerosis, low-density lipoprotein receptor–deficient (Ldlr^−/−) mice fed with high cholesterol diet were treated with either Reelin ASO or neutralizing antibody (CR-50) to systemically deplete circulating Reelin. In both treatments, atherosclerotic plaque progression was markedly attenuated. These in vivo results suggest that Reelin depletion decreases vascular adhesion and inhibits the recruitment of monocytes and consequently prevents plaque progression.

Conclusions:

These findings suggest that Reelin inhibition may provide a novel therapeutic approach to counteract leukocyte or monocyte adhesion as well as extravasation and inhibit the progression of atherosclerosis. This strategy may also be relevant for other diseases that involve leukocyte or monocyte extravasation as a central pathological mechanism, such as multiple sclerosis or arthritis.

Graphical Abstract

INTRODUCTION

Endothelial dysfunction has been associated with the inflammatory process underlying the development of atherosclerosis ¹. In endothelial cells (EC), the Apolipoprotein E receptor-2 (Apoer2 or Lrp8), a low-density lipoprotein receptor (LDLR) family member, is involved in inflammatory processes such as nitric oxide synthesis and regulation of endothelial cell surface expression of leukocyte adhesion molecules ^1–4. Systemic Apoer2 deficiency has been associated with increased susceptibility to atherosclerosis in mouse models ^5,6, and in human genomic studies ^7–9. However, Apoer2 deletion in mice has also been demonstrated to have anti-thrombotic effects in the endothelium by reducing leukocyte-endothelial cell adhesion in response to antiphospholipid antibodies ^10,11. We sought to better understand the cell-specific effects of Apoer2 in atherosclerosis by focusing on the effect of the Apoer2 ligand Reelin ⁴ in endothelial cells.

Originally described in the brain, Reelin guides neurons to their correct positions in the developing brain and modulates synaptic function in the adult brain ^12–16. Outside the nervous system, we have previously shown that circulating Reelin promotes vascular adhesion and leukocyte infiltration by elevating the basal state of vascular immune surveillance ^4,17. In the atherosclerosis-prone LDLR-deficient (Ldlr−/−) mouse model, Reelin was deleted ubiquitously or only in the liver, thus preventing its release into the circulation. In these two models, Reelin deletion reduced leukocyte-endothelial adhesion as well as the expression of the pro-inflammatory markers vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). This was accompanied by decreased atherosclerosis progression and macrophage content in atherosclerotic lesions. It was confirmed in human endothelial cell (EC) cultures that Reelin increased monocyte adhesion as well as the expression of ICAM1, VCAM1, and E-selectin. Interestingly, Reelin action was mediated by its receptor Apoer2, followed by upregulation of nuclear factor κB (NF-κB) activity.

Together, these previous findings demonstrate that the Reelin-Apoer2 signaling cascade regulates leukocyte adhesion on the endothelium, thus promoting chronic inflammation and atherosclerosis progression. To lay the foundation for future therapeutic interventions, we tested whether reducing circulating Reelin, using antisense oligonucleotide (ASO) or neutralizing antibodies, would limit atherosclerotic plaque progression in the atherosclerosis-prone Ldlr^−/− mouse model.

MATERIALS AND METHODS

Authors will make their data, analytic methods, and study materials available to other researchers upon request.

Cell culture.

Commercially available human aortic endothelial cells (HAEC; ATCC; PCS-100-011) were cultured in EBM-2 Medium (Lonza; CC-3156) supplemented with EGM-2 (Lonza; CC-4176) on petri-diches coated with gelatin. Commercially available human aortic smooth muscle cells (HASMC; ATCC; PCS-100-012) were cultured in SmGM-2 BulletKit medium (Lonza; CC-3182) for smooth muscle cells as previously described ^18–20. The monocyte cell line U937 (human histiocytic lymphoma; CRL-1593; ATCC) was grown in RPMI 1640 medium (Sigma-Aldrich) containing 10% FBS. For in vitro experiments, all cells were used between passages 3 and 8.

Protein expression:

Cells were serum starved (1% fetal bovine serum (FBS)) overnight and then stimulated as described in the figure legends with doses tested in previous studies ^4,17,18. Then, cells were washed and protein harvested in RIPA buffer.

shRNA transduction:

shRNA transduction was performed as previously described ¹⁸. shRNA lentivirus against Dab2 was purchase from Origen (TL313573).

Monocyte adhesion assay:

HAEC were seeded and grown in 6-well plate to form a confluent monolayer. Cells were serum starved (1% FBS) overnight and then stimulated as described in the figure legends, with the following components for 24 hours: human plasma (dilution 1/50 in starvation medium), human plasma depleted for Reelin with CR-50 immunoprecipitation (Dr. Katsuhiko Mikoshiba originally provided the CR-50 hybridoma). Then the cells were washed with PBS and incubated with human monocyte (U937) for 45min at 37°C with gentle rocking. Finally, the cells were washed and fixed with PFA 4%. The number of adherent monocytes was manually counted in 5 random areas per well, with at least six wells per condition.

Cx3cr1-GFP mice and Intravital microscopy for quantification of monocyte–endothelial cell adhesion.

Cx3cr1-GFP mice (B6.129P-Cx3cr1^tm1Litt/J) were purchased from The Jackson Laboratory (Stock No. 005582). These mice express EGFP in monocytes, dendritic cells, NK cells, and brain microglia under the control of the endogenous Cx3cr1 locus. Cx3cr1-GFP monocytes downregulate GFP expression upon differentiation into macrophages.

For intra-vital microscopy, 4-week-old Cx3cr1-GFP males and females received one injection (25mg/kg, intra-peritoneally) of control or anti-Reelin ASO. Live imaging was then performed one week later.

After anesthesia with Ketamine/xylazine, the mesentery of 5-week-old Cx3cr1-GFP mice was exposed for observation and recording of images of GFP-monocyte adhesion and rolling using a Regita digital camera (QImaging) attached to a Nikon Eclipse Ti microscope with NIS Element image-capturing software. Per mice, videos on 6 different marginal mesenteric veins were recorded. The velocity and quantity of monocyte rolling were measured by ImageJ (wrmtrck plugin) on each video for 10 seconds. The rolling index was calculated as monocyte number / velocity for each record. For each 10-second-video, all the frames were stacked into one final picture and the intensity was measured to visually reflect and measure the monocyte exposition to the vascular wall.

After recording, the mice were sacrificed, blood was collected for white blood cell count and plasma analysis, the thoracic aorta was dissected out, the adventitia was removed and the media-intima was snap frozen for further protein analysis.

Cx3cr1-GFP; Ldlr^−/− mice and atherosclerosis model.

Cx3cr1-GFP line was crossed with Ldlr^{−/− 21} to obtain the Cx3cr1-GFP; Ldlr^−/− mice. To evaluate atherosclerosis severity, 8-week-old male and female mice were placed on an atherogenic high-cholesterol diet containing 21% (w/w) milk fat, 1.25% (w/w) cholesterol, and 0.5% (w/w) cholic acid (TD 02028, Harlan Laboratories), for 16 weeks.

Mice euthanized by anesthetic overdose and hearts were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde, and hearts and the entire aorta were collected. For en face analysis, entire aortas from the heart extending 5 to 10 mm beyond the bifurcation of the iliac arteries were removed and dissected free of adjoining tissues, opened, and stained with Oil Red O. Lesion extent was evaluated by morphometry of scanned images using ImageJ software.

Western blot.

Cell lysates or tissue pieces were prepared by adding protease and phosphatase inhibitor cocktail in RIPA buffer and centrifuging for 10 minutes at 12000 rpm to removed debris. From the lysates, protein concentrations were determined by the Lowry protein assay (500-0113, 500-0114, 500-0115; Bio-Rad). Equal amounts of protein were loaded into each lane of a 4-12% Tris gel (BioRad) and subjected to electrophoresis. After blotting, nitrocellulose-membranes (BioRad) were blocked for 1h (milkpowder 5% in TBS/tween 0.1-0.2%) and incubated overnight at 4°C with primary antibodies (pPI3K, Abcam, ab12135; PI3K, Upstate, 05-212; pAkt, Biosource, 44-622Z; Akt, Cell Signaling, 9272S; pIκBα, Abcam, ab12135; pP65, Cell Signaling, 3036S; P65, Cell Signaling, 4764S; pSTAT3, Cell Signaling, 9131; STAT3, Cell Signaling, 9139; E-Selectin, Santa Cruz, sc-137054; ICAM-1, R&D Systems, AF796; G10 anti-Reelin, made in house; Reelin, Millipore, MAB5366; GAPDH, Sigma-Aldrich, G8795), all diluted at 0,2 μg/ml. Binding of secondary HRP-antibodies were visualized by ECL or ECL plus chemiluminescent (Amersham). After densitometric analyses with ImageJ, optical density values were expressed as arbitrary units and normalized for protein loading to the total form of a protein (for the phosphorylated proteins) or to a housekeeping protein as indicated on the figures, as described in the figure legends.

RT-qPCR.

Total RNA was extracted according to the TRIzol protocol (TRIzol, Life Technologies). RNA concentrations were measured spectrophotometrically (Nanopdrop 2000c; Thermo Scientific). For RNA, first strand cDNA was synthesized using SuperScript III enzyme according to the manufacturer’s instructions (11752; Life Technologies). Quantitative PCR reaction was then performed with TaqMan probes (E-selectin, IDT, Hs.PT.58.1165629; ICAM-1, IDT, Hs.PT.58.4746364; VCAM-1, IDT, Hs.PT.58.20405152, B2M, IDT, 58v.18759587). For each sample, multiplexed reaction was performed, for the target gene (with FAM reporter) and the housekeepers B2M or RPS9 (with VIC reporter). Quantitative PCR reaction was performed with TaqMan^TM probes using the TaqMan^TM Universal Master Mix II (4440046; Life Technologies) according to the manufacturer’s instructions. Relative quantity (RQ) values were calculated using the ViiA7 software V1.2.2 (Applied Biosystems).

Statistics.

For cell culture, each condition was tested at least in duplicate (unless specified differently), and all experiments were repeated at least 3 times at different passages. For animals, the “n” values are specified in each legend. The software GraphPad Prism was used to run all the statistical analysis. Values from multiple experiments are expressed as mean±SEM. Normality was tested using the Kolmogorov-Smirnov test. Statistical significance was determined for multiple comparisons using one-way analysis of variance (ANOVA) followed by Turkey multiple comparison (for normal distribution) or Kruskal-Wallis followed by Dunn multiple comparison (for non-normal distribution) test. Student’s t-test (for normal distribution) or Mann Whitney (for non-normal distribution) were used for comparisons of two groups. Equal variance was tested. p<0.05 was considered significant.

RESULTS

Reelin signaling in HAEC promotes monocyte adhesion

In neurons, Reelin binds to Apoer2 to induce phosphorylation of the intracellular adapter protein Disabled-1 (Dab1), which then drives activation of PI3K/Akt, followed by IKK and IκBα and NF-κB ^22,23. We hypothesized that a similar signaling cascade exists in HAEC, but through Dab2 instead of Dab1 due to the predominance of this variant in endothelial cells.

Stimulation of HAEC with Reelin resulted in first increased phosphorylation of PI3K, Akt and IκBα, followed in time by NF-κB (Figure 1A). In addition, Reelin promoted the mRNA expression of adhesion molecules E-selectin, ICAM-1 and VCAM-1 which was inhibited by the depletion of Dab2 using an shRNA (Figure 1B). These results confirmed in HAEC that Reelin triggers a phosphorylation cascade similar to that observed in neurons that involves Dab2, PI3K, Akt and NF-κB to promote the expression of adhesion markers.

Figure 1. Reelin signaling in HAEC promotes monocyte adhesion.

(A) In a time-course experiment, HAEC were starved and stimulated with Reelin (20nM) for the indicated time to look at phosphorylation cascade (PI3K, Akt, IκBα, NFκB) by western blot. Representative blots are shown (n=6, ANOVA / Kruskal-Wallis). (B) In expression experiment, HAEC were transduced using shRNA negative control (NC) or targeting Dab2 mRNA (Dab2). Then, cells were starved and stimulated with Reelin (20nM) for 24 hours to look at mRNA expression of DAB2 and adhesion markers (E-selectin, ICAM-1, VCAM-1) by RT-qPCR (n=3, ANOVA). (C) Human plasma was depleted by immunoprecipitation using protein G beads with mouse IgG (control antibody) or CR-50 (anti-Reelin antibody), to obtain “normal” (=N) and “Reelin-depleted” (=D) plasma respectively, and a representative blot is shown. In adhesion functional assay, HAEC were starved and stimulated with PBS as control or human plasma (“normal”=N or “Reelin-depleted”=D) for 1 or 24 h. After washing, the cells were incubated with human monocytes (U937) for 45 min, washed and fixed. Adherent monocytes appearing as small white round cells were counted, and representative pictures are shown (n=6, Kruskal-Wallis). *p<0.05 and **p<0.01.

Next, we investigated the role of Reelin in monocyte adhesion to HAEC in an in vitro system. Human plasma was immunoprecipitated with protein G beads bound to either mouse IgG or CR-50, a mouse anti-Reelin antibody, to obtain “normal” and “Reelin-depleted” plasma, respectively (Figure 1C). HAEC were then incubated with human plasma followed by exposure to human monocytes. Treatment with human “normal” plasma induced monocyte adhesion on endothelial cells at 1 and 24 hours, while the “Reelin-depleted” plasma induced significantly less monocyte adhesion and was similar in effect to untreated HAEC.

Together, these experiments demonstrate in HAEC that Reelin-Apoer2 activates the PI3K / Akt / NF-κB signaling pathway, promotes the expression of adhesion molecules and consequently induces adhesion of monocytes to endothelial cells.

Reelin promotes HASMC proliferation via Stat3

In contrast to canonical (NF-κB) signaling recruited in HAEC, we have also observed a strong phosphorylation of Stat3 induced by Reelin in HASMC. After treatment of HASMC with Reelin, Stat3 phosphorylation peaked at 2 hours, while total Stat3 expression was not affected (Figure 2A). Stat3 is known to regulate cell proliferation in many cell types including SMC. We therefore investigated the role of Reelin/Stat3 on HASMC proliferation using a crystal violet staining assay (Figure 2B). As depicted in Figure 2B, Reelin-induced HASMC proliferation was inhibited by both CR-50 and the Stat3 inhibitor (BP-1-102). These data suggest that reelin, through Stat3 activation, is mitogenic in HASMCs.

Figure 2. Reelin promotes HASMC proliferation via Stat3.

(A) Phosphorylated and total protein expression of Stat3 was evaluated by western blot in HASMC (n≥6, ANOVA). (B) Proliferation was assessed by cell count on HASMC fixed and stained with crystal violet. Cells were starved overnight and incubated as indicated with Reelin (20nM), CR50 (100nM) and Stat3 inhibitor (BP-1-102; 5μM) for 72h. Scale bar = 200μm and representative pictures are shown (n≥6, ANOVA / Kruskal-Wallis). *p<0.05 and **p<0.01.

Anti-Reelin Treatment decreases endothelial adhesion of monocytes

We have previously demonstrated that genetic depletion of Reelin on an Ldlr−/− background impedes monocyte rolling on the vessel wall ⁴. To explore in vivo the potential of pharmaceutical Reelin depletion on monocyte rolling, we tested anti-Reelin ASO treatment in intravital microscopy. A Cx3cr1-GFP mouse model with genetically GFP-labelled monocytes was used. Seven days after a single ASO intraperitoneal injection, the number of rolling monocytes attached to the endothelial surface was greatly reduced in the setting of reelin depletion, and rolling velocity was increased compared to control (Figure 3A). This resulted in decreased monocyte interaction with the vascular wall as shown by decreased rolling index and cumulative intensity (illustrated by representative cumulative pictures; Figure 3A). We did not observe any significant differences in the monocyte adhesion or velocity between male and female mice. White blood cell number and the vessel area measured by intravital microscopy was similar in both ASO groups.

Figure 3. Anti-Reelin ASO inhibits adhesion of monocytes to endothelial cells.

(A) 4-week-old Cx3cr1-GFP male and female mice were injected intraperitoneally with control (n=10) or anti-Reelin ASO (n=10) at 25mg/kg. Intravital microscopy was performed 7 days after injections to record numbers and speed of rolling monocytes on the marginal mesenteric vessels. 6 different vessels / mouse were recorded for 10 sec and analyzed; cumulative pictures represent individual images (100 frames/second) integrated over a 10-second period and stacked together; rolling index = monocyte number / velocity; t-test / Mann Whitney. (B,C) After intravital microscopy, immunoblotting was performed on plasma and aorta protein extracts. *p<0.05 and **p<0.01 (t-test/Mann Whitney).

We confirmed that Reelin was effectively depleted in the plasma of Anti-Reelin ASO treated mice (Figure 3B) and that this correlated with decreased expression of rolling (E-selectin) and adhesion (ICAM1) markers in the aorta (Figure 3C). This is consistent with our previously published findings showing that Reelin increases E-selectin and ICAM-1 expression in endothelial cells ^4,17. These data support Reelin as a therapeutic target for the inhibition of monocyte adhesion and recruitment, with potential impact for perivascular inflammation.

Anti-Reelin Treatment decreases plaque progression in atherosclerosis

Interaction of Reelin with Apoer2 ^24–29, and Apoer2 itself play a central role in atherosclerosis development, inducing vascular cell adhesion molecule expression and leukocyte–endothelial cell adhesion ^1–3. Therefore, we hypothesized that pharmacological Reelin blockade would dampen atherosclerosis progression. To test this hypothesis, Cx3cr1-GFP; Ldlr^−/− mice were treated with ASO targeting Reelin mRNA or CR-50 antibody targeting Reelin protein, both effectively decreasing circulating Reelin (Figure 4A,D) and initiated with the high-cholesterol feeding.

Figure 4. Anti-Reelin interventions attenuate atherosclerotic lesion development in Ldlr^−/− mice.

8-week-old Ldlr^−/− male and female mice were fed with high-cholesterol diet for 16 weeks. Littermates were randomly distributed in four groups and treated with 25mg/kg control ASO (n=11) versus anti-Reelin ASO (n=12) (A-C), or with 100 μg control IgG (n=11) versus anti-Reelin antibody (CR-50; n=11) (D-F). (A,D) 16 weeks after starting the high-cholesterol feeding, plasma Reelin was measured by western blot (Mann Whitney). (B,E) Representative en face photomicrographs and average percent of aortic lesion area stained with Oil Red O are presented (t-test). (C,F) Effect of 16 weeks of high cholesterol diet on the average body weight, fasting plasma cholesterol and triglyceride concentrations are presented. *p<0.05 and **p<0.01 (t-test / Mann Whitney).

After high-cholesterol feeding for 16 weeks beginning at 8 weeks of age, aortas were excised and stained with Oil Red O to visualize atherosclerotic plaques. En face analyses revealed a decrease in atherosclerotic lesion area in aortas from mice treated with Anti-Reelin ASO or antibody compared to control mice treated with control ASO or IgG respectively (Figure 4B,E). Notably, control versus Reelin-depleted mice (either with ASO or CR-50) had similar body weights, and plasma cholesterol and triglyceride concentrations (Figure 4C,F), consistent with our previous results in the systemic and liver-specific Reelin deficient mouse models ⁴. Taken together, our results show that Reelin depletion by both ASO and CR-50 greatly diminished atherosclerotic lesion formation, independent of plasma lipoprotein levels.

DISCUSSION

In this study, we demonstrated that reduction in circulating Reelin either by ASO or neutralizing antibody CR-50 markedly attenuated atherosclerotic plaque progression in Ldlr^−/− mice fed with high cholesterol diet. In vivo, ASO treatment effectively decreased leukocyte-endothelial adhesion as shown by intravital microscopy, suggesting that by decreasing vascular adhesion, anti-Reelin treatment inhibits the recruitment of monocytes and consequently prevents plaque progression. Using human plasma in vitro to study Reelin mechanism, we demonstrated that untreated plasma induced monocyte adhesion to HAEC, while Reelin-depleted plasma had no effect on adhesion. In addition, signaling analysis in HAEC revealed that Reelin activated a Dab2, PI3K, Akt and NF-κB cascade to promote the expression of adhesion markers (E-selectin, ICAM-1, and VCAM-1). Finally, in HASMC, Reelin induced Stat3 phosphorylation to promote cell proliferation, which is a hallmark of plaque progression.

These results are in accordance with and expand our previous studies on Reelin contribution in the development of chronic inflammatory diseases ^4,17. We have previously shown in the atherosclerosis-prone Ldlr^−/− mouse model ⁴, and in a mouse experimental autoimmune encephalomyelitis model for multiple sclerosis ¹⁷, that Reelin deletion protected against monocyte recruitment and infiltration. Specially, we studied atherosclerosis-prone Ldlr−/− mice in which we deleted Reelin either ubiquitously or only in the liver, thus preventing the production of circulating Reelin ⁴. In both types of Reelin-deficient mice, atherosclerosis progression was markedly attenuated, and macrophage content and endothelial cell staining for VCAM-1 and ICAM-1 were reduced at the sites of atherosclerotic lesions. In cultured human endothelial cells, Reelin enhanced monocyte adhesion and increased ICAM1, VCAM1, and E-selectin expression by increasing NF-κB activity in an Apoer2-dependent manner. These findings demonstrate that genetical depletion of circulating Reelin protects from atherosclerosis by decreasing vascular inflammation. Here, we show similar protection with two pharmacological anti-Reelin treatments, the ASO and the antibody CR-50 administrated by intraperitoneal injection. While both result in a drastic diminution of Reelin concentration in plasma, they have different modes of action. ASO abolishes Reelin synthesis, which is abundantly produced by stellate cells in the liver ^30–33, by entering into cells and degrading Reelin mRNA. On the other hand, CR-50 binds to circulating Reelin protein and thus, clears it most likely through classical immunocomplex degradation.

The atherosclerosis plaque characterization was performed in our previous study ⁴. Briefly, we investigated the presence of macrophages in atherosclerotic plaques by immunostaining. Morphometric quantification of the lesions revealed a reduction of Mac-3–positive macrophages in Reelin KO mice compared to Ldlr−/− control mice ⁴. To determine the impact of Reelin genetical deletion on macrophage accumulation in the lesions, we measured VCAM-1 and ICAM-1 abundance in plaques by immunostaining. Both were reduced in endothelium overlying plaques in Reelin KO mice. These results suggest that Reelin promotes macrophage foam cell accumulation in atherosclerotic lesions, and that this is likely driven by increased adhesion molecule expression.

In addition, here we detail the phosphorylation cascade in HAEC, by showing the activation of Dab2, PI3K and Akt, in addition to NF-κB, as previously demonstrated ⁴. This cascade highlighted in HAEC is in accordance with previous studies in neurons and confirms phosphorylation of similar mediators ^34–36. Of note, unlike in neurons, in HAEC, we have not observed Erk phosphorylation under Reelin stimulation. Another difference concerns the Dab protein adaptor for Apoer2. In the nervous system, Reelin binds to Apoer2 and to very low-density lipoprotein receptor (Vldlr), leading to phosphorylation of the intracellular adapter protein Dab1 by Src-family tyrosine kinases ^27–29,37, which then activates PI3K/Akt ^28,38, thereby controlling various cellular functions, including neuronal positioning, synaptic plasticity, and memory formation ³⁹. In endothelial cells, instead of Dab1 we focused on Dab2, which is believed to be more relevant as an Apoer2 adaptor protein in endothelial cells, whereas Dab1 is predominant in neurons ⁴⁰.

Finally, we report a previously unknown effect of Reelin on vascular SMC (VSMC) proliferation. Apoer2 deficiency in SMCs has been associated with cell cycle arrest and phenotypic shift from a proliferative to a secretory phenotype ⁶. It has been shown that Apoer2 deficiency inhibits the formation of the PP2A-C – CDC20 complex, which prevents activation of the APC/C-CDC20 complex, which is required for progression through the cell cycle. Here, we confirmed that Apoer2-mediated signaling induced VSMC proliferation, however through a different mechanism involving Reelin-induced phosphorylation of Stat3. Consequently, Reelin depletion was also associated with decrease in cell proliferation. This mechanism is especially relevant for atherosclerosis since VSMC phenotypic switch (from contractile to proliferative synthetic cells) and aberrant proliferation with intimal invasion promotes plaque formation ^41,42. However, the detailed pathway activated by Reelin in VSMC remains unclear. Indeed, the Reelin receptor Apoer2 is abundantly expressed in the vascular wall cells, including platelets, monocytes/macrophages, endothelial cells, and SMC ⁴³. It has been described in multiple myeloma that Reelin activates Stat3 via integrin α5β1 (specially via the subunit β1) ^44,45, integrin which is also expressed on VSMC membrane ⁴⁶. Interestingly, direct Reelin interaction with integrins could also explain how Reelin promotes short term (1 hour) monocyte/endothelial cell adhesion without de novo synthesis of additional adhesion molecules (Figure 1C). The novel, Stat3-mediated signaling observed in VSMC would require a dedicated study to investigate the recruitment of integrins.

In conclusion, we have exploited a function of Reelin in the regulation of leukocyte-endothelial adhesion to devise a novel strategy for combating inflammation and plaque progression in atherosclerosis. Despite the broad clinical use of statins to lower LDL cholesterol, atherosclerosis is still an important cause of cardiovascular morbidity and mortality. This underlines the need for adjunctive non-lipid-lowering therapies against the progression of atherosclerotic disease. Reelin depletion decreases atherosclerosis progression independently of lipid levels, and therefore supports targeting the inflammatory side of atherosclerosis, which could be an adjunct to the widely accepted lipid-lowering therapies. Beyond cardiovascular diseases, anti-Reelin strategies would be expected to be similarly effective for the treatment of other chronic inflammatory syndromes that depend upon excessive leukocyte extravasation such as multiple sclerosis, arthritis or Crohn’s disease.

Figure 5. Therapeutic Reelin depletion decreases endothelial adhesion of leukocytes and protects against atherosclerosis.

This mechanistic model incorporates the findings described in this article using pharmacological depletion of Reelin, our previous article using genetical depletion of Reelin ⁴, and the literature to date as discussed. In vascular endothelial cells, Reelin promotes the expression of vascular adhesion proteins, thereby increasing monocyte adhesion / extravasation, inflammation and consequently plaque progression. In vascular smooth muscle cells, Reelin promotes cell proliferation via Stat3 phosphorylation. Therapeutic Reelin depletion prevents this inflammatory cascade and thus atherosclerosis. We surmise that these mechanisms are conserved in humans.

HIGHLIGHTS SECTION.

• Pharmacological Reelin depletion (either by ASO or neutralizing antibody) decreases plaque progression in atherosclerosis mouse model.

• Reelin via Apoer2 activates Dab2, PI3K, Akt and NF-κB cascade to promote the expression of adhesion markers (E-selectin, ICAM-1, and VCAM-1) in vascular endothelial cells and therefore, Reelin promotes monocyte/endothelial cell adhesion.

• Identification of a non-canonical Reelin pathway via Stat3 activation to promote vascular smooth muscle cell proliferation.

Abbreviations

EC

endothelial cells

Apoer2 or Lrp8

Apolipoprotein E receptor-2

LDLR

low-density lipoprotein receptor

VCAM-1

vascular cell adhesion molecule-1

ICAM-1

intercellular adhesion molecule-1

NF-κB

nuclear factor κB

ASO

antisense oligonucleotide

HAEC

human aortic endothelial cells

FBS

fetal bovine serum

PBS

phosphate-buffered saline

Dab1

Disabled-1