Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction.

Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed in vivo during disease is unknown. In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation.

Introduction

Inflammation is a carefully orchestrated response involving the release of pro-inflammatory factors and homing of immune cells to injured tissue. The vascular endothelium is a key player in the inflammatory process, responding to stressors such as oscillatory blood flow (that occurs at bifurcations in the vessel) and increased pro-inflammatory factors [1-3]. The resulting activated endothelium provides a pro-adhesive surface that allows circulating immune cells to adhere and migrate into the inflamed tissue in a multistep process of capture, rolling, firm adhesion, and transmigration. These steps are mediated by multiple endothelial surface adhesion molecules [4-9].

Many surface and secreted proteins, including adhesion molecules, are N-glycosylated. While this post (or co-)-translational modification regulates protein stability and transport to the cell surface, these glycan structures are also important in mediating binding between cognate receptors, which is exemplified by binding between selectins and their sialyl LewisX ligands [10, 11]. Relatively little is known, however, about how N-glycosylation of endothelial adhesion molecules may be regulated during inflammation [12]. Protein N-glycosylation occurs in the endoplasmic reticulum-Golgi network via a multi-step process involving initial attachment of a core oligosaccharide, comprised of Glu3Man9GlcNAc2, onto the amide residue of asparagine within the consensus sequence N-X-S/T (where X cannot be proline). Sugars from this core are temporally and sequentially cleaved by ER/Golgi resident glycosidases, leading to high mannose structures, followed by partial rebuilding to hybrid N-glycan structures (collectively referred herein as hypoglycosylated N-glycans) by glycosyltransferases; after which multiple fucose, GlcNAc, and sialic acid residues are added to form complex N-glycoproteins [13]. While it is assumed that most glycoproteins require complete processing to the complex form for cell-surface expression, we and others have demonstrated that during atherogenic inflammation, the endothelial surface is characterized by decreased N-glycan complexity, or an increase in hypoglycosylated N-glycans [11, 14–16]. However, the exact endothelial proteins that harbor these hypoglycosylated N-glycans and their presence in vivo remains unclear.

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that mediates leukocyte adhesion and whose expression is induced by pro-inflammatory stimuli. ICAM-1 has 8 putative N-glycosylation sites and we have previously shown that, in activated cultured endothelial cells, both high mannose/hybrid and complex (specifically α2,6-sialylated) ICAM-1 can be expressed on the cell-surface [15]. Interestingly, we observed that hypoglycosylated ICAM-1 N-glycoforms bound monocytes with higher affinity and regulated “outside-in” signaling via different mechanisms compared to ICAM-1 glycoforms containing complex, α-2,6-sialylated N-glycans. Furthermore, non-classical pro-inflammatory (CD14+CD16+) monocytes, but not classical (CD14+CD16-) monocytes, selectively recognize HM-ICAM-1 to roll and adhere to activated endothelial cells [15, 17, 18]. These findings suggest unique and distinctive functions for the different ICAM-1 N-glycoforms. However, whether these distinct ICAM-1 N-glycoforms are present in vivo is not yet known.

Herein, we tested whether different ICAM-1 N-glycoforms are present in vivo by modifying the DuoLink® proximity ligation assay (PLA) to detect co-localization of specific N-glycan structures and ICAM-1 in two disease states where endothelial dysfunction has been demonstrated to play a prominent role (see for assay schematic and lectin specificity). Both human and mouse atherosclerotic tissue, as well as arteriovenous fistulas (AVF) of advanced chronic kidney disease patients, were evaluated for the presence of ICAM-1 N-glycoforms. Our data demonstrate that HM-ICAM-1 N-glycoforms are present in advanced disease states at similar, if not higher, levels compared to α-2,6-sialylated N-glycoforms. Importantly, HM-ICAM-1, but not α-2,6-sialylated ICAM-1 positively correlated with macrophage burden as assessed by CD68 staining suggesting a significant role for hypoglycosylated N-glycans in vascular inflammation.

Proximity-ligation assay (PLA) schematic and lectin specificity.

Biotinylated lectins with indicated specificities were added to first label specific sugars. Then, anti-ICAM-1 antibody and avidin, conjugated to complementary oligos, were added. When lectin-recognized sugar epitopes are less than 40 nm from anti-ICAM-1, the complementary oligos hybridize and amplify, producing red fluorescent puncta. Right panel shows N-glycan structures recognized by different lectins used (adapted from [19]. Man = Mannose; Gal = Galactose; GlcNac = N-acetylglucosamine; Sa = Sialic acid; R = varying N-glycan structures.

Materials and methods

Materials

DuoLink® proximity ligation assay kit was purchased from ThermoFisher (Waltham, MA). HistoPrep™ was purchased from Fisher Scientific (Hampton, NH). Anti-human ICAM-1 antibody was purchased from ThermoFisher (BMS108), and anti-mouse ICAM-1 antibody was purchased from Abcam (25375) (Cambridge, UK). Concanavalin A (ConA), Sambucus Nigra (SNA), Hippeastrum Hybrid Amaryllis (HHL), and Maackia Amurensis Lectin II (MAL-II) lectins were purchased from Vector laboratories (Birlingame, CA). Anti-ICAM-1 and avidin were conjugated to oligomer probes using the DuoLink Probemaker kits (Sigma) per manufacturer provided protocols. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Human vessel collection and processing

Human specimens were collected at the University of Alabama at Birmingham or Louisiana State University Health Sciences Center according to procedures and protocols approved by the University of Alabama at Birmingham Institutional Review Board and Louisiana State University Health Sciences Center Institutional Review Board respectively. Human arteries were obtained post-mortem, after authorization of autopsy that includes permission to remove tissues for research purposes. Vessel segments were fixed using formalin (10%) for 24h and then embedded in paraffin. Lesion type was determined according to the Stary scale from 1–5 [20] by a cardiovascular pathologist using the defining characteristics of fibrosis, calcification, and fatty deposits. All procedures were performed per Institutional Review Board approved protocols.

In addition, human vein samples were collected from subjects at the time of 1st and 2nd stage basilic vein transposition surgery. At the time of both surgeries, a circumferential piece of vein (~10–15 mm and adjacent to site of anastomosis creation) was excised and immediately stored in formalin for histology and immunohistochemistry (IHC) studies. Each venous tissue sample was embedded in paraffin as previously described [21]. Following paraffin embedding, each piece was sliced into 5μM sections for histological studies. These vein samples were collected under approval by the University of Alabama at Birmingham Institutional Review Board. Donor specimens from cadaveric donors at the time of organ harvesting were obtained using services from the National Disease Research Interchange, Philadelphia, PA, and fixed in formalin, as previously described [22].

Mouse vessel collection and processing

8–10 week old male ApoE-/- mice were fed a high fat, Western-type diet (21% fat by weight, 0.15% cholesterol, and 19.5% casein without sodium cholate) for 12 weeks, and the innominate artery (i.e. brachiocephalic) and left carotid arteries collected for histological analysis. Both lesion and non-lesion areas of the same segment were analyzed and lesions identified by the presence of increased neointimal hyperplasia compared to non-lesion segments. Carotid arteries were also collected from male ApoE-/- mice fed a control diet. In the second model, 8–10 week old, male ApoE-/- mice underwent partial ligation of the left carotid artery to induce disturbed flow as described previously [19, 20]. After induction of anesthesia (5% isoflurane, followed by 2% isoflurane for maintenance of anesthesia), the external, internal, and occipital arteries were ligated with a 7–0 silk suture leaving the superior thyroid artery patent, and the resulting disturbed flow pattern was verified by ultrasound measurements using a VisualSonics VEVO3100 system. When this model is performed in atherosclerosis-prone ApoE-/- mice, accelerated atherosclerosis develops in the left carotid artery due to disturbed flow compared to the paired right carotid artery [21]. Mice were euthanized by pneumothorax under isoflurane anesthesia, and then ex-sanguinated followed by vessel collection. For all samples, the adventitia was carefully removed and vessel segments fixed in formalin (10%) for 24h and then embedded in paraffin blocks for mounting to slides. All procedures were performed according to LSU Health Sciences Center-Shreveport and University of Alabama at Birmingham IACUC approved protocols.

Tissue immunofluorescence

Paraffin-embedded serial tissue sections were rehydrated using standard protocols. Briefly, slides were treated with xylene followed by decreasing Histoprep concentrations (100%, 95%, 85%, 75%) before immersion in 1x PBS. Antigen retrieval was accomplished using 10mM sodium citrate buffer (pH 6.0) and heated for 12 minutes in the microwave. Following antigen retrieval, tissues were washed in PBS and proximity-ligation, total ICAM-1, or CD68 staining performed as outlined below.

Proximity-ligation assay

Briefly, 20 μg of the following biotinylated lectins: HM and hybrid N-glycan-specific lectin ConA, the α2,6-sialyation specific lectin SNA, the HM-specific lectin HHL, or the α2,3-sialylation specific lectin MAL-II were added for 30 minutes at 4°C (see ). These lectins were chosen to provide broad coverage of different N-glcyan structures. After washing twice with PBS, samples were blocked with 1X Carbo-Free blocking solution (Vector Labs) for 30 min at 20–25°C. Immediately following blocking, samples were incubated with oligo-tagged avidin or anti-ICAM-1 (10 μg/mL) for one hour at 37°C followed by ligation and amplification steps as per the protocol. Both anti-mouse and human ICAM-1 antibodies recognize extracellular (domain 1) epitopes on ICAM-1. Slides were left to dry and mounted using the DuoLink® mounting medium containing DAPI.

Total ICAM-1 and CD68 immunofluorescence

Slides were processed as described above. After blocking, slides were incubated with mouse or human anti-ICAM-1 or CD68 O/N at 4°C, followed by a 1 hour incubation at 20–25°C with the species-appropriate secondary antibody conjugated to Alexa Fluor 594. After washing, slides were left to dry and mounted using DuoLink® mounting medium containing DAPI.

Image acquisition and analysis

Images were acquired on a BioTek Lionheart live cell imager using DAPI (377/447), GFP (469/525), and Texas Red (586/647) filters. LED intensity, camera gain, and integration time for each channel was uniform across all samples. Images at three random locations within lesion and non-lesion areas were recorded per section. Images were collected at 4x and 60x magnification. Image quantification was performed using either Gen5 (Biotek) or ImageJ software (NIH). To quantify PLA images, 3 random fields per group, per experiment were selected and number of puncta counted using ImageJ particle analysis software (NIH).

Statistics

All statistical analysis was performed using GraphPad Prism software. Paired student t-tests were utilized in comparing the different areas (lesion vs. non-lesion) from the same vessel or left and right carotids from the same animal. Unpaired t-test was used to compare human vessels with different lesion stages. A p-value less than 0.05 was considered significant.

Results

Hypoglycosylated ICAM-1 is present in mouse atherosclerotic lesions

Vessels from ApoE-/- mice were collected after 12 weeks feeding of a control or high fat diet, with the latter being an established method to reproducibly promote atherosclerosis [7, 22, 23]. Total ICAM-1 levels (measured using an anti-ICAM-1 antibody that binds to epitopes on the N-glycan-devoid domain 1 of the protein) were detected on the luminal surface of lesions, consistent with prior reports of up-regulation of this adhesion molecule on the endothelium [24-26] (). No ICAM-1 was detected on areas of the vessel devoid of lesions, nor on vessels collected from mice fed a normal chow diet. To determine which N-glycoforms of ICAM-1 were present, we used the PLA assay (), where red puncta indicate positive staining that the two epitopes are within 40nm of each other. To measure HM and hybrid ICAM-1, samples were labeled with biotinylated ConA, followed by treatment with avidin and an anti-ICAM-1 antibody both tagged with complementary oligonucleotides. High mannose N-glycans, α-2,6-sialylation, and α-2,3-sialylation on ICAM-1 were measured using the same method with HHL, SNA and MAL-II lectins, respectively. shows red puncta for the ICAM-1 N-glycoforms in lesion areas only; no PLA staining was observed in non-lesion areas of the same vessels nor in vessels from ApoE-/- mice fed normal chow (control diet). As an immunostaining control, the antibody against ICAM-1 or avidin were excluded ( respectively) and showed no PLA-positive staining associated with lesions. shows that total ICAM-1 and HM / hybrid N-glycoforms of ICAM-1 were significantly increased in lesion vs. non-lesion regions, with similar trends with α-2,6-sialylated ICAM-1 noted (p<0.07). Macrophage burden was assessed by CD68 staining in lesion and non-lesion regions; these measurements were performed on serial sections from the same preparations used for proximity ligation assay staining. CD68 staining was significantly higher in lesion areas compared to non-lesion areas (). Note: Since different lectins will bind their target epitopes with distinct affinities, direct comparison between lectins is not possible.

HM epitopes co-localize with ICAM-1 in high fat-induced mouse atherosclerosis.

Total, HM / hybrid, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 were measured in the innominate and left carotid arteries from ApoE-/- mice fed a normal or high fat diet. A) Shown are representative images of innominate arteries from paired lesion and non-lesion areas of the same vessel section. Red staining represents total ICAM-1, red puncta represent positive PLA staining for specific ICAM-1 N-glycoforms (indicated by arrows), and blue staining represents DAPI. # indicates the lumen of each vessel. Panels B and C show PLA staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded. Panel D shows total ICAM-1 staining in lesion versus non-lesion areas. *p<0.05 by t-test. Panel E shows number of puncta for HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 in lesion versus non-lesion areas. Data are mean ± SEM, each symbol represents a distinct mouse, n = 6. *p<0.05 compared to non-lesion via paired t-test. F. Representative images of CD68 staining (red) in lesion vs. non-lesion areas. G. Quantitation of CD68 staining in lesion and non-lesion areas. Data are mean ± SEM, each symbol represents a distinct mouse, n = 6. * = p<0.05 compared to non-lesion via paired t-test. ● from innominate arteries and ○ from left carotid arteries.

We also collected vessels from ApoE-/- mice that underwent partial carotid ligation, which creates oscillatory flow in the left carotid artery while leaving the right carotid artery as an internal control. This model rapidly causes endothelial dysfunction and atherosclerosis [19]. Both the left and right carotid arteries from mice that underwent partial ligation were collected 7 days post-ligation and stained for total ICAM-1 and ICAM-1 N-glycoforms. show representative images and quantification of ICAM-1 and ICAM-1 N-glycoform staining. Total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). CD68 staining was also significantly higher in the LC compared to the control RC ().

HM / hybrid, HM, and α-2,6-sialylated ICAM-1 are increased in mouse atherosclerosis after induction of disturbed flow in vivo.

Panel A shows representative images of total ICAM-1 HM / hybrid, HM, α2,6-sialylated, and α2,3-sialylated ICAM-1 in the left carotid artery (after partial ligation) and paired right carotid artery (control). Positive PLA puncta are indicated by arrows. Panels B & C show total ICAM-1 staining in left versus right carotid artery and ICAM-1 N-glycoforms puncta, respectively. Data are mean ± SEM, each symbol represents an individual mouse, n = 3. *p<0.05 compared to RC via paired t-test. D. Representative images of CD68 staining (red) in LC vs. RC areas in mice. E. Quantitation of CD68 staining in LC and RC areas. Data are mean ± SEM, n = 3. *p<0.05 compared to RC via t-test. # indicates the lumen of each vessel.

show correlations between CD68 staining and HM / hybrid, HM, and α-2,6-sialylated, ICAM-1 respectively, from ApoE-/- mice fed high fat diets and after partial carotid ligation. Significant positive correlations were observed between CD68 and HM / hybrid and HM-ICAM-1 in both mouse models, but only HFD-fed mice had a significant positive correlation between CD68 and α-2,6-sialylated, ICAM-1.

CD68 macrophage staining positively correlates with HM-ICAM-1 in animal models of atherosclerosis.

A-C. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice fed HFD diet. n = 6 animals; n = 12 paired lesion and non-lesion vessel areas. D-F. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice after partial carotid ligation n = 3 animals; n = 6 vessel samples. Best fit lines determined by linear regression with Pearson correlation analyses with indicated coefficients and p-values shown in each panel.

HM / hybrid and α2,6-sialylated ICAM-1 are present in advanced human atherosclerotic lesions

To determine whether distinct ICAM-1 N-glycoforms are present in human atherosclerosis and whether they were dependent on disease severity, human vessels were collected at autopsy and luminal surface total ICAM-1 and HM / hybrid, α2,3-sialylated, and α2,6-sialylated ICAM-1 were measured via PLA. summarizes patient demographics, vessel location, and lesion type. ICAM-1 expression increased as a function of disease severity with staining visible in type 3 and 5 lesions (). Similarly, HM-/ hybrid, α-2,6-sialylation, and α-2,3-sialylation N-glycoforms were only detected in advanced lesions () – show specificity for HM / hybrid and sialylated-ICAM-1 staining; no PLA-positive staining was observed when individual PLA reagents were omitted.

Fig 5

HM / hybrid ICAM-1 is increased in human atherosclerosis.

Panel A shows representative images of total ICAM-1 (red staining) and specified N-glycoforms in human vessels with lesions spanning types 1–5. Red puncta represent positive PLA staining (as indicated by arrows). Panel B shows the quantification of total ICAM-1 in early (1–2) and late (3–5) disease stages. Each symbol represents a different patient, with same symbol representing multiple vessels from the same patient. Data are mean ± SEM, n = 7–10. * p<0.05 compared to types 1–2 via unpaired t-test. Panel C shows the quantification of HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 puncta in early (1–2) and late (3–5) disease stages. * = p<0.05 compared to early stage lesions by t-test. Data are mean ± SEM. n = 7–10. Panels D and E show staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded.

Finally, CD68 staining was significantly higher in advanced lesions compared to earlier stage lesions () or compared to IgG control staining of advanced lesions. Further, CD68 staining had a significant positive correlation with HM-ICAM-1 levels with a trend towards significance (P<0.09) noted for correlation between α-2,6-sialyated ICAM-1 and CD68 levels. ().

CD68 macrophage staining positively correlates with HM / hybrid ICAM-1 in human atherosclerosis.

Panels A and B show representative images and quantitation, respectively, of CD68 staining in advanced and early type lesions, as well as IgG control staining. Data are mean ± SEM, n = 7–10 * p<0.05 compared to types 1–2 via unpaired t-test. C-E. ConA, HHL, and SNA puncta plotted against CD68 staining. Data are from n = 17 vessel samples collected from 12 subjects; each symbol represents a different subject. Best fit lines were determined by linear regression analyses indicated coefficients determined by Pearson correlation analyses.

HM / hybrid and α2,6-sialylated ICAM-1 are present in patients on hemodialysis with failed AVFs

Advanced chronic kidney disease (CKD) patients that reach end-stage renal disease require hemodialysis. A common limitation with this therapy is failure of the AVF to successfully mature due to endothelial dysfunction and inflammation mediated in part by oscillatory flow [27, 28]. Basilic vein samples from CKD patients that had undergone surgical AVF creation were obtained and subjected to PLA for HM/hybrid, α-2,3-sialylated, and α-2,6-sialylated ICAM-1. shows patient demographics, and the success vs. failure status of AVF. show representative images indicating that total ICAM-1, HM / hybrid, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 were present in the failed AVFs. Little to no ICAM-1 was detected in successful AVFs (. HM / hybrid and α-2,6-sialylated ICAM-1 levels were significantly higher in failed AVFs compared to successful AVFs, with α-2,3-sialylated ICAM-1 also being elevated (p<0.06) (). No staining for ICAM-1 or associated N-glycoforms (only tested with ConA and SNA) were observed in control (no AVF) healthy donor veins (n = 3).

HM / hybrid, HM, α-2,6-sialylated, ICAM-1 are increased CKD patients with failed arteriovenous fistulas.

Panel A shows total ICAM-1 (red staining) and specified N-glycoforms in vessels from CKD patients with failed or successful AVF creation. Red puncta represent positive PLA staining (as indicated by arrows). B) Quantification of total ICAM-1 signal in failed and successful AVF samples (n = 4 each). Error bars are mean ± SEM; * p<0.05 compared to successful AVF samples by t-test. C) Quantification of HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 as puncta per total ICAM-1 signal. Error bars are mean ± SEM; * p<0.05 compared to successful AVF samples by unpaired t-test.

Discussion

Inflammation is a carefully orchestrated process involving the homing of immune cells to inflamed tissues, a process mediated by adhesion molecules on the endothelial cell surface [5]. Surface adhesion molecules are heavily N-glycosylated with ~20–50% of their observed molecular weights attributed to N-glycans. While insights into the role of protein N-glycosylation in regulating inflammation have primarily focused on circulating immune cells (e.g. CD44 and other selectin ligands on neutrophils and T-cells [10,21,29], relatively little attention has been paid to the impact N-glycans may have on the function of endothelial adhesion molecules in diseases mediated by endothelial dysfunction.

ICAM-1 is a well-established mediator of increased monocyte-endothelial interactions and interestingly, compared to the related adhesion molecule, VCAM-1, contains more N-glycans as a proportion of its overall molecular weight (~50% in ICAM-1 vs. ~25% in VCAM-1) [30]. Numerous studies have shown that deletion of ICAM-1 or blocking its ability to engage Mac-1 or LFA-1 on leukocytes prevents inflammation [31-34]. It is also known that N-glycosylation can modulate ICAM-1 function. For example, Mac-1 binding is higher to ICAM-1 bearing HM structures[35]. Moreover, different ICAM-1 N-glycoforms may regulate tumor burden and inflammatory signaling, and that the degree of ICAM-1 N-glycosylation can change depending on the cell in which it is expressed [36-40]. In our previous work, we showed that HM / hybrid ICAM-1 was formed in activated endothelial cells, and this hypoglycosylated ICAM-1 N-glycoform mediated higher affinity binding to monocytes compared to fully processed, sialylated ICAM-1 N-glycoforms. In addition, ligation dependent interactions between ICAM-1 and the actin cytoskeleton was also distinctly regulated between HM / hybrid ICAM-1 and complex-ICAM-1[15,30].

While the aforementioned studies suggest altered functions for hypoglycosylated vs. complex sialylated ICAM-1, whether these N-glycoforms are expressed in disease is not known and important to establish translational significance. By using vessels from patients with varying stages of atherosclerotic lesions, from CKD patients with successful or failed AVFs, and from two atherosclerotic mouse models (ApoE-/- mice fed high-fat diet and ApoE-/- mice post-partial carotid ligation), we show that HM / hybrid and complex ICAM-1 N-glycoforms are found in vascular lesions but not in non-lesion areas. Interestingly, HM / hybrid ICAM-1 levels displayed stronger correlations with CD68 macrophage staining in human and mouse lesions, suggesting that hypoglycosylated ICAM-1 may play a more prominent role in mediating monocyte recruitment compared to α-2,6-sialyated ICAM-1. Consistent with this hypothesis, our previous data showed that the affinity of monocyte adhesion to HM / hybrid ICAM-1 was greater compared to α-2,6-sialyated ICAM-1[18,30].

To date, very few studies have focused on N-glycosylation of endothelial cells and their role in disease. The complexity of studying surface adhesion proteins and their N-glycan patterns may be a contributing factor to the lack of studies in this area. For example, ICAM-1 contains 8 putative N-glycosylation sites. This, coupled with the different possible combination of N-linked sugars ranging from HM- to complex N-glycans, means that ICAM-1 may exist in >200,000 possible N-glycoforms (calculated from data in Lau et al [41]. This complexity clearly makes identification of specific N-glycoforms challenging [17,42]. It is for this reason we employed 4 different lectins in this study to identify different ICAM-1 N-glycoforms. By using lectins that recognize high-mannose, hybrid, and two different types of sialic acid linkages, we detected ICAM-1 decorated with at least 4 different types of N-glycans in vivo (Note: ConA and HHL have some overlapping N-glycan binding specificities). To our knowledge, this data represents the first study demonstrating distinct N-glycoforms on ICAM-1 in vivo. We do note limitations of our study, namely the sole reliance on the PLA assay to discern HM, hybrid, and sialylated-ICAM-1 and that this assay only informs on spatial proximity; it is possible that HM-epitopes on other proteins and ICAM-1 are co-expressed. However, our previous data show that ICAM-1 immunoprecipitated from human atherosclerotic lesions has a similar molecular weight (75KDa) to HM / hybrid ICAM-1; complex ICAM-1 is close to 100KDa, supports conclusions that a HM-ICAM-1 N-glycoform is present in vivo [15]. An additional limitation is quantitation of PLA-positive puncta. Differential affinities for binding between specific N-glycan structures and the different lectins could lead to different staining intensities. For this reason, we quantified data based only on the number of positive puncta and not on staining intensity. However, we recognize that relative differences in lectin binding may have affected sensitivity for detecting discreet PLA-positive puncta. Potential limitations in detection also preclude definitive conclusions regarding temporal expression of ICAM-1 and its N-glycoforms as a function of disease. Type 2 lesions are characterized by lipid laden macrophages; however neither ICAM-1 protein, nor its N-glycoforms, were detectable in these lesions in our methods. ICAM-1 expression in early atherosclerosis has been studied, and in some cases has been found to be minimal in earlier stages of the disease [43,44], which may explain the lack of detection in our methods. Further, the role of ICAM-1 in early atherosclerosis is still debated [45], and our data suggests this HM-ICAM-1 may be important in later stages of the disease.

Further studies using more specific and selective approaches for N-glycan analyses are required to determine the exact N-glycoforms of ICAM-1 present in vascular inflammation and their function. Little is known about the regulation and control of different N-glycoforms, and if the regulation may be disease specific. For example, while our data showed little α-2,3-sialylated ICAM-1 in human atherosclerosis, it was present in failed AVFs from humans. This observation perhaps indicates that regulation of N-glycoforms depends on the disease state. Future studies in the lab will focus on the regulation of N-glycans during inflammation; specifically by looking at the enzymes responsible for N-glycosylation, such as α-mannosidases and glycosyltransferases.

Distinct biological functions of ICAM-1 N-glycoforms may also have therapeutic implications. Attempts to therapeutically target ICAM-1 to abrogate atherosclerosis in vivo have been successful in animal models; for example, ICAM-1 deficiency and anti-ICAM-1 antibody treatment protects against atherosclerosis in ApoE-deficient mice [34,46]. However, anti-ICAM-1 therapies have not translated into humans. Treatment with functional blocking anti-ICAM-1 antibodies have had no effect in improving kidney allograft rejection rates nor mortality rates in stroke patients, and in fact induced a neutrophil-dependent pro-inflammatory response in stroke patients [47-49]. Moreover, a general concern with a long-term anti ICAM-1 therapeutic strategy is the potential for inhibiting innate immunity [50]. ICAM-1 is a diverse protein with functions spanning mitogenic signaling, leukocyte adhesion, and cell survival mechanisms [51-53], and therefore global targeting can result in a potentially detrimental disruption of normal responses. Our findings that different N-glycoforms of ICAM-1 are expressed, coupled with these having distinct functions [15,18], may offer new therapeutic strategies that involve targeting hypoglycosylated HM-ICAM1 specifically. We posit that blocking HM / hybrid ICAM-1 may lead to selective attenuation of monocyte ingress into atheroprone endothelial beds. A better understanding of the glycosylation patterns of the activated endothelium throughout the course of disease may yield more selective therapeutics that target endothelial inflammation in a disease and vascular bed-specific manner.

29 Aug 2019

PONE-D-19-20764

Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction

PLOS ONE

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Reviewer #1: The study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan structures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients (models of endothelial dysfunction). The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF.

1. The figures of the manuscript are very low resolution.

2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta.

3. How were the lesion and non-lesion segments identified?

4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments.

5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies.

In the high fat diet model (Fig. 2; no ligation), there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation.

6. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al (ATVB, 1995). These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels.

7. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracy.

8. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data.

9. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different.

Reviewer #2: This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis.

To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay.

There was no description for Figure 2F and 2G in result section. Author should mention about these two figures.

In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy.

In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes.

Partial ligation and HFD are well establish model for atherosclerosis. Authors used only male mice without justification for the exclusion of female mice. Author should comment on this in discussion.

Overall, this manuscript does not fit for publication in PLoS One as it currently stands.

Reviewer #3: The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger.

1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study.

2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well.

3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence.

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5 Feb 2020

Dear Dr. Fukai,

Thank you for forwarding reviewer comments on our article “Assessment of ICAM-1 N-glycoforms in Mouse and Human Models of Endothelial Dysfunction”. We appreciate the reviewer’s constructive comments. In the revised manuscript, we have addressed the comments raised. Specific responses are provided below with edits in the text indicated by red font color.

Thank you

Sincerely

Rakesh Patel, PhD

Reviewer #1:

The study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan stuctures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients (models of endothelial dysfunction). The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF.

1. The figures of the manuscript are very low resolution.

Author response: Image resolution has been increased. Al images are at 600dpi resolution per journal guidelines.

2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta.

Author response: We did not have access to mouse aortic tissue, but have now included new data from the left carotid arteries from ApoE-/- mouse fed high fa diet. These vessels are commonly used to interrogate atherosclerosis disease mechanisms; see revised Fig 2.

3. How were the lesion and non-lesion segments identified?

Author response: Human lesions were characterized by a cardiovascular pathologist using the defining characteristics of fibrosis, calcification, and fatty deposits. Mouse lesion segments were identified by the presence of increased neointimal hyperplasia compared to non-lesion segments.

4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments.

Author response: We apologize for this oversight. The manuscript has been updated with additional experiments and n numbers corrected accordingly.

5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies.

Author response: Additional replicates have been added and correlation analyses separated out into HFD-fed experiments and partial carotid ligation to promote clarity.

6. In the high fat diet model (Fig. 2; no ligation), there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation.

Author response: As explained above, additional experiments were performed. With these additional replicates and the separation of models in the correlation analysis, ConA and HHL had a significant positive correlation with CD68 staining in HFD-mice and partial carotid ligation.

7. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al (ATVB, 1995). These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels.

Author Response: We do not make the claim that ICAM-1 N-glycoforms play a role in early monocyte adhesion. However, the lack of ICAM-1 staining may also be due in part to detection limitations in our assay. We have included discussion of this point in the revised manuscript.

8. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracy

Author response: Additional data was added to manuscript and legends adjusted accordingly.

9. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data.

Author response: Recently published work out of our lab demonstrates that different ICAM-1 N-glycoforms selectively recruit monocyte subsets under flow. Please see citation # 18 in the manuscript.

10. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different.

Author response: Mechanisms leading to AVF do indeed differ from atherosclerosis, but endothelial dysfunction as a result of turbulent blood flow is an underlying component of both diseases states. We wanted to test distinct ICAM-1 N-glycoforms were also present in other disease settings where vascular inflammation and turbulent blood flow are underlying features. This is also further discussed in the manuscript.

Reviewer #2:

This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis.

1. To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay.

Author response: Image resolution has been increased.

2. There was no description for Figure 2F and 2G in result section. Author should mention about these two figures.

Author response: We apologize for this oversight. The manuscript has been edited to include these figures in the results section.

3. In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy.

Author response: Analysis of PLA puncta is between paired LC and RC sections, not across lectins (i.e., α-2,6-sialylated ICAM-1 vs. HM/hybrid ICAM-1). Direct comparison between lectins is not possible as each have different binding affinities to their target epitopes- similar to how two different antibodies may bind with distinct affinities. The presented data is to show an increase in the ICAM-1 N-glycoforms in the ligated vessel compared to the non-ligated vessel (LC vs RC). We have clarified this point in the text.

4. In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes.

Author response: Correlation studies were performed by measuring CD68 staining in the same (serial) sections on which PLA staining was performed. In the case of HM and hybrid ICAM-1, CD68 staining increased as puncta increased, indicating a positive association between the two. Statistical analyses were performed using Pearson correlation.

Reviewer #3:

The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger.

1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study.

Author response: Image resolution has been increased.

2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well.

Author Response: By western blot analyses, we have shown previously that ICAM-1 immunoprecipitated from atherosclerotic lesions has a MWt close to 75KDa suggesting it is a HM-glycoform. Our published studies show that ICMA-1 with complex N-linked sugars has a MWt closer to 100KDa. This information is included in the revised discussion.

3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence.

Author Response: Immunofluorescence has been used extensively to assess CD68 staining in lesions, and per our experience there are no pros/cons in IF vs IHC for this marker. We have added an IgG staining control to Figure 6 to further verify specificity of the antibodies used for IF-based detection of CD68.

Submitted filename: PLoS response final.docx

Click here for additional data file.

28 Feb 2020

Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction

PONE-D-19-20764R1

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Reviewer #3: Revised manuscript has been improved, since image quality has been improved and necessary information was added. No further comments.

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3 Mar 2020

PONE-D-19-20764R1

Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction

Dear Dr. Patel:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Table 1

Atherosclerosis patient demographics (N.D. = no data).

Patient #	Age	Sex and Race	Atherosclerotic Lesion Type	Vessel Sample(s)
1	28	Female, White	12	Right coronaryCircumflex
2	47	Male, White	2	Right coronary
3	22	Male, N.D.	3	Left anterior descending
4	22	Male, African American	3	Right coronary
5	66	Male, African American	455	CircumflexLeft anterior descendingRight coronary
6	58	Male, White	2	Thoracic Aorta
7	31	Male, White	1	Thoracic Aorta
8	64	Female, White	53	Left coronaryCircumflex
9	45	Female, White	2	Aorta
10	55	Female, White	2	Left anterior descending
11	58	Male, African American	43	Left anterior descendingLeft coronary
12	78	Male, White	3	Right coronary

Table 2

AVF patient demographics.

Patient #	AVF Status	Age	Sex and Race	Vessel Sample(s)
13	Successful	55	Male, White	Basillic
14	Successful	77	Female, African American	Basillic
15	Successful	54	Male, White	Basillic
16	Successful	72	Female, African American	Basillic
17	Failed	73	Female, African American	Basillic
18	Failed	63	Female, African American	Basillic
19	Control–no AVF	58	Female, White	Basillic
20	Control–no AVF	69	Female, White	Basillic
21	Control–no AVF	56	Female, White	Basillic
22	Failed	58	Female, African American	Basillic
23	Failed	36	Male, African American	Basillic