CD82 Restrains Pathological Angiogenesis by Altering Lipid Raft Clustering and CD44 Trafficking in Endothelial CellsCLINICAL PERSPECTIVE
Background—Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown.
Methods and Results—Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44.
Conclusions—Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.
Vascular morphogenesis includes vasculogenesis and angiogenesis.1 Both involve coordinated endothelial cell (EC) proliferation, EC migration, branching, and tube formation.2 Deregulated vascular morphogenesis and pathological angiogenesis contribute to the pathogenesis and progression of diseases ranging from cancer and macular degeneration to chronic inflammation.3 Growth factors promote neovascularization,4 whereas cell adhesion molecules (CAMs) are also crucial for vascular morphogenesis.2 More important, signals from growth factors and CAMs cross-talk during vascular morphogenesis.5,6 For example, integrin αvβ3 interacts with growth factor receptors and plays complex roles in angiogenesis.6,7
Clinical Perspective on p 1504
Tetraspanins regulate cell adhesion, migration, fusion, and proliferation.8 Tetraspanin CD82 modulates immune cell activation and viral infection and suppresses tumor progression. In migrating cells, CD82 overexpression inhibits both protrusive and retractive cellular processes by disrupting actin reorganization.9 CD82 overexpression also alters cell adhesions.9 At the plasma membrane, CD82 interacts with membrane lipids such as GM2 and modulates membrane lipid composition.10–12 CD82 inhibits integrins, EGFR, c-Met, and urokinase-type plasminogen activator receptor9 and reduces downstream signaling of Src, p130CAS/Crk, Rho small GTPases, and β-catenin.9 However, how CD82 regulates cytoskeletal organization and membrane protein activities is still unclear.
CD82 is expressed in ECs and arteriolar smooth muscle cells,13–15 but whether it regulates vascular function remains unknown. We found that pathological angiogenesis and EC movement were increased in Cd82 knockout (KO) mice, likely resulting from the upregulations of CAMs and their initiated signaling. Such upregulations were caused by sequential changes in gangliosides, lipid rafts, CAM-membrane microdomain interactions, and then CAM endocytosis. Hence, CD82 modulates CAM trafficking by preventing lipid raft aggregation and dissociating CAMs from lipid rafts, and CD82-ganglioside-CD44 signaling restrains angiogenesis by inhibiting EC adhesiveness and motility.
Reagents, Polymerase Chain Reaction, and Cellular Function Assays
Detailed descriptions of reagents, polymerase chain reaction (PCR) analyses, and cell migration, invasion, sprouting, adhesion, proliferation, and survival assays are given in the online-only Data Supplement.
Mice and Cells
The establishment of the CD82-null mouse line, mouse genotyping strategy, and isolation of primary ECs are described in the online-only Data Supplement. Animal studies were performed with approval from the institutional animal care and use committees.
Detailed descriptions of the in vivo Matrigel plug, tumor, retina, myocardial infarction (MI) angiogenesis, and ex vivo aortic ring angiogenesis are given in the online-only Data Supplement.
Confocal Microscopy, Fluorescence Resonance Energy Transfer, and Total Internal Reflection Fluorescence Microscopy
See the online-only Data Supplement for details.
Stochastic Optical Reconstruction Microscopy
ECs were plated on fibronectin-coated MatTek dishes for 24 hours and then incubated with Alexa 647–conjugated cholera toxin subunit B for 20 minutes on ice, washed, and fixed. Stochastic optical reconstruction microscopy imaging was performed as described previously.16 Briefly, image acquisition was performed on a Nikon Eclipse Ti microscope with a 150-mW, 647-nm laser in total internal reflection fluorescence mode on continuous illumination. Thirty thousand frames per image were collected at a rate of 50 Hz with a ×100 PlanApo 1.45NA Nikon objective projected on an Andor iXon DU897 electron-multiplying charge-coupled device camera. Single-molecule fitting and image rendering were performed with N-STORM software within NIS Elements (version AR 4.13.04) with a localization precision of ≈40 nm.
See the online-only Data Supplement for details.
Data are presented as mean±SEM or mean±SD and were analyzed with JMP pro 11 software (SAS Institute Inc, Cary, NC). The normality of data was examined before any test. For 2-group comparisons, 2-tailed, unpaired Student t tests were performed if samples exhibited normal distribution, and nonparametric Wilcoxon rank-sum tests were performed if samples were not normally distributed. For multiple-group comparison, Kruskal-Wallis tests followed by Dunn tests were performed if the Kruskal-Wallis test was significant. Differences are considered significant for values of P<0.05.
Endothelial CD82 Regulates Vascular Morphogenesis
To confirm Cd82 expression in endothelium, we examined CD82 mRNA in murine primary ECs isolated from lung and liver by droplet digital PCR and found that Cd82 was expressed in these ECs. The level of CD82 mRNA was almost 3 times higher than the level of hypoxanthine-guanine phosphoribosyltransferase, a housekeeping gene (Figure IA in the online-only Data Supplement). In addition, we used flow cytometry to detect CD82 proteins at the surfaces of human dermal microvascular ECs, human umbilical vein ECs, and human retinal capillary ECs. We found that CD82 was expressed in these human ECs (Figure IB in the online-only Data Supplement).
Then, we examined CD82 vascular function using a gene ablation approach. The Cd82 KO mouse line was generated by the use of homologous recombination and Cre-LoxP deletion strategies (Figure IIA in the online-only Data Supplement). Because Cre is driven by human cytomegalovirus promoter and gene deletion occurs at all cells,17 the deletion of Cd82 gene was expected to be ubiquitous and was confirmed by Southern blotting and PCR at the DNA level and quantitative real-time PCR at the mRNA level in various tissues (Figure IIB and IIC in the online-only Data Supplement). For example, using quantitative real-time PCR, we found that the levels of the truncated CD82 mRNA in mouse lung or liver ECs (MLECs) from Cd82-null mice varied from 3% to 6% of the levels of the full-length CD82 mRNA from WT mice (Figure IID in the online-only Data Supplement). The Cd82-null mice are viable and fertile in C57BL/6 and FVB genetic backgrounds.
To determine the role of CD82 in vascular morphogenesis, we first performed Matrigel plug angiogenesis assay. The Matrigel plugs excised from Cd82-null mice exhibited a marked increase in neovascularization, as determined by immunofluorescent and immunohistochemical analyses of CD31 expression (Figure 1A). Vascular area and microvessel density increased by ≈100% and 130%, respectively, upon Cd82 ablation. More blood perfusion, which is correlated with functional vessel formation and evidenced by color and hemoglobin content of the Matrigel plugs, was found in Cd82-null mice compared with wild-type (WT) mice. Increased lumen formation was proportional to more vasculature in the Cd82-null group, suggesting that CD82 is dispensable for proper tubulogenesis. CD31 staining was always surrounded by and partially colocalized with the staining of the pericyte marker NG2, confirming that CD31 labels vasculature. Second, we examined tumor angiogenesis by subcutaneously implanting syngenic Lewis lung carcinoma cells. In the implanted tumor, angiogenesis was dramatically greater in Cd82-null mice than in WT mice (Figure 1B), as was tumor size.
For retinal angiogenesis at P18, by which active angiogenesis is largely completed, WT and Cd82-null mice displayed no obvious difference in forming superficial radial and collateral vessels, suggesting that CD82 is not required for the ultimate development of retina vessels (Figure IIIA in the online-only Data Supplement). For hyperoxia-induced ablation of retinal blood vessels, which leads to an avascular area, no difference was observed between the WT and KO groups (Figure IIIB in the online-only Data Supplement). After mice are returned to normoxia, the surge of neovascularization resulting from the vessel ablation forms vessel tufts to alleviate ischemia. The tuft formation was profoundly higher in both number and area in Cd82-null mice than in WT mice (Figure 1C), further supporting that CD82 preferentially inhibits pathological retinal angiogenesis.
MI induces active angiogenesis during the acute phase. Angiogenesis in the MI regions of Cd82-null mice at 1 week after MI was apparently more pronounced than in the WT mice (Figure 1D). Importantly, the recovery of cardiac function, reflected by the ejection fraction of left ventricle, was also significantly better in Cd82-null than the WT group.
To substantiate these findings, we examined the ex vivo ability of the aortic artery to undergo angiogenesis. Cd82-null aortic rings had greater microvascular sprouting, with the length and area of sprouted vessels increasing by ≈58% and 64%, respectively (Figure 1E). BS1-lectin staining confirmed that the sprouts were endothelial.
ECs assemble into capillary networks in 3-dimensional extracellular matrixes, which mimics vasculogenesis. We found that the cable network formation was also markedly enhanced in Cd82-null MLECs after ECs were seeded in fibrin gel for 2, 8, and 24 hours (Figure 1F and Figure IV in the online-only Data Supplement). Time-lapse videomicroscopy revealed that EC migration leading to cable formation was increased in Cd82-null ECs compared with WT ECs (data not shown).
CD82 Mainly Alters EC Migration and Invasion
To address the cellular mechanism by which CD82 restrains pathological angiogenesis, we assessed the roles of CD82 in EC proliferation and survival. Cd82 removal slightly promoted EC proliferation and survival (Figure V in the online-only Data Supplement).
Because CD82 inhibits cell movement in cancer cells,9 we also examined cell movement. First, significantly greater migration was detected for Cd82-null ECs onto fibronectin or laminin 111, toward chemoattractants, than for WT ECs (Figure 2A). Second, in vitro invasion of ECs through Matrigel was also markedly greater in the Cd82-null than the WT group (Figure 2B). More important, the depth that newly formed vessels penetrate into Matrigel gel plug was much greater in Cd82-null than in the WT group (Figure 2B), suggesting that Cd82-null ECs also exhibited higher invasiveness in vivo. Third, the penetration distance of ECs from their coated beads in fibrin gel was largely enhanced on CD82 silencing (Figure 2B). Because mouse ECs hardly attach to the beads, we performed this experiment with human umbilical vein ECs. Furthermore, markedly more CD82-silenced ECs were found at the tips of endothelial sprouts originating from the beads coated with equal numbers of CD82-silenced and nonsilenced ECs (Figure 2C), indicating that ECs gained invasiveness on CD82 reduction. Notably, changes in cell migration and invasion are apparently much larger than those in cell proliferation and survival.
Because tetraspanins regulate microextrusion morphogenesis and microextrusions may modulate cell movement,18 we examined microextrusion in ECs. Compared with WT ECs, Cd82-null ECs formed more and developed longer CD31- and CD44-containing microextrusions (Figure 2D), supporting that CD82 inhibits microextrusion morphogenesis.
During retinal angiogenesis, tip cells and their filopodia were markedly increased at P5 in Cd82-null compared with WT retinas (Figure IIIC in the online-only Data Supplement), strongly suggesting that more invasive and robust vessels are developed during angiogenesis in Cd82-ablated mice.
Because CD82 associates with CAMs, we examined cell-matrix adhesion. Cd82-null MLECs showed marked increases in adhesion onto hyaluronan and laminin 111 but no change onto fibronectin (Figure 2E).
CD82 Inhibits the Cell Surface Presence of Endothelial CAMs
To determine how CD82 regulates EC motility and adhesiveness, we examined the effect of Cd82 ablation on EC surface expression of tetraspanins and CAMs by using flow cytometry. Tetraspanin CD9, integrin α6, integrin αV, and CD44 were upregulated in Cd82-null ECs, whereas others remained equivalent between 2 groups (Figure 3A). Because CD44 associates with tetraspanins and plays roles in EC motility and angiogenesis,19–22 we compared CD44 protein and mRNA levels in WT and Cd82-null ECs and found that total CD44 proteins became increased upon Cd82 ablation (Figure 3B) but CD44 mRNA remained unchanged (Figure 3C). Similarly, the upregulated surface level of integrin α6 was not accompanied by an increase in integrin α6 mRNA level (Figure 3C). These observations suggest that the increases in CD44 and integrin α6 protein levels on Cd82 ablation were not a result of altered gene transcription but were due to changes in protein turnover. Such a conclusion is consistent with no correlations in gene expression between CD82 and CD44 or integrin α6 in endothelia, although a reverse correlation exists between CD82 and integrin αV genes (Figure VI in the online-only Data Supplement).
EC adhesion onto hyaluronan, a matrix ligand of CD44, was enhanced without CD82 (Figure 2E), reflecting an increase in functional CD44 proteins. In addition, CD44 was upregulated in the vessels of the Matrigel plug implanted in Cd82-null mice (Figure 3D). Moreover, CD44 monoclonal antibody reduced aortic ring angiogenesis and endothelial network formation of Cd82-null ECs to the WT level (Figure 3E and 3F and Figure VII in the online-only Data Supplement). Furthermore, CD44 monoclonal antibody inhibited EC migration and reduced the migration of Cd82-null ECs to the level of WT ECs (Figure 3G).
To substantiate this finding, we examined CD44 expression in MI-induced angiogenesis (Figure VIIIA in the online-only Data Supplement). In heart tissue from normal rats, CD44 expression was negligible in capillary within myocardium, indicating that CD44 is minimally expressed in quiescent microvessels. At weeks 1 and 2 after MI, massive angiogenesis was accompanied by a drastic increase in endothelial CD44. At week 4 after MI, CD44 expression was significantly reduced in ECs within the MI areas that underwent fibrosis. Thus, CD44 expression in ECs is correlated with angiogenesis. The same conclusion can be reached during the angiogenesis after MI in WT and Cd82-null mice (Figure VIIIB in the online-only Data Supplement).
CD82 Restrains Cytoskeletal Connection and Signaling of CAMs
The changes in CAMs drove us to investigate their cytoskeletal connection. In Cd82-null ECs, tetraspanins CD9 and CD81 were localized in focal complex–like structures at the cell periphery (Figure 4A). Integrin β1 exhibited unaltered distribution but formed more focal adhesions in CD82-null ECs (Figure 4B). Higher staining intensities of integrin α6 and CD44 were found in CD82-null ECs, but their global cellular distributions appeared to be unchanged (Figure 4B and 4C).
Focal adhesion formation and development were markedly enhanced in Cd82-null ECs on the basis of the staining of talin, a marker of nascent focal adhesion, and paxillin and vinculin, constituents of focal adhesion (Figure 4D).
FAK/Src-p130CAS/Crk signaling is regulated by CAMs and tetraspanins and controls cell movement.2,9 The protein level and autoactivation (pY397) of FAK remained unchanged, but full activation of FAK (pY577) was elevated in Cd82-null ECs (Figure 4E). Tyr410-phosphorylated p130CAS and Tyr416-phosphorylated c-Src were markedly greater on Cd82 ablation, indicating an upregulated FAK/Src-p130CAS signaling axis.
PI3K-Akt signaling, denoted by Ser473-phosphorylated Akt, was upregulated in Cd82-null ECs (Figure 4F). ERM proteins (Figure 4G), which link tetraspanins and CD44 to actin cytoskeleton, and vascular endothelial growth factor receptor-2 (Figure 4H), which triggers angiogenic signaling, remained unaltered.
CD82 Modulates CD44 Endocytosis and Lipid Raft Clustering
Because tetraspanins regulate endocytosis,8,9 more CAMs at the Cd82-null EC surface may result from less endocytosis. Indeed, absolute amounts of the internalized CD9 and CD44 were lower in Cd82-null MLECs than in WT MLECs after 60 minutes of endocytosis (Figure 5A and Figure IXA in the online-only Data Supplement). After normalization to their levels at the cell surface, CD9 and CD44 internalization dropped further in the KO group. In contrast, the endocytosis of CD81 and integrin α5, the levels of which were not altered on Cd82 ablation, was equivalent between the 2 groups. Earlier studies showed that CD44 is internalized through clathrin-independent endocytosis pathway.23,24 Using GM1, a cargo of clathrin-independent endocytosis pathway route, as tracer, we found that most internalized CD44 proteins colocalized with GM1 in both groups after 2 and 5 minutes of endocytosis (Figure 5B and Figure IXB in the online-only Data Supplement), suggesting that Cd82 ablation did not alter the endocytosis route of CD44. However, CD44 and GM1 internalizations were substantially reduced after 5 minutes of endocytosis in Cd82-null ECs (Figure 5B and 5C), suggesting that clathrin-independent endocytosis of CD44 and this endocytic pathway per se were both inhibited without CD82.
Because CD82 overexpression alters the interaction between tetraspanin-enriched microdomains (TEMs) and lipid rafts,12 we analyzed the distributions of CD44, the TEM marker CD9, and the lipid raft marker GM1 at the EC basal surface with total internal reflection fluorescence microscopy. CD44 distributions were similar in a majority of ECs (Figure 6A) but displayed a clustered pattern in approximately one third of Cd82-null ECs (Figure 6A, middle). CD9 exhibited similar staining characteristics at the basal surface between the 2 groups. Notably, GM1-positive lipid rafts were evenly distributed to a large extent in fixed WT cells but became clustered much more often to form small patches in fixed KO cells. Using super-resolution imaging, we confirmed that GM1 frequently formed clusters with sizes of ≈30 to 50 nm at the basal surface of Cd82-null ECs. Normalized Ripley K function [L(r)-r] determines whether a given pattern is clustered, random, or dispersed.25 Positive humps in the normalized curves indicated clustering over those distances. Cd82-null ECs displayed significant clustering of GM1 over short distances compared with WT cells. Latrunculin dispersed CD44 and GM1 clusters (Figure X in the online-only Data Supplement), suggesting actin involvement in clustering and excluding GM1 clusters as postfixation artifacts.
We then examined the colocalizations of CD44 with TEM and lipid raft markers at the basal section (an ≈230-nm-thick focal plane) of fixed MLECs with confocal microscopy (Figure 6B and Figure XI in the online-only Data Supplement). The colocalizations of CD44 with GM1- or flotillin-positive lipid rafts and CD9-positive TEMs were significantly increased in Cd82-null ECs (Table), suggesting that more CD44 is coalescent with TEMs and rafts.
Using fluorescent probes C16Dil and C16DiO, which incorporate to lipid-ordered regions at the plasma membrane,26 we found that the fluorescence resonance energy transfer signal from C16Dil and C16DiO (Figure XII in the online-only Data Supplement) was drastically elevated in Cd82-null ECs and that such an elevation could be abrogated by the raft disrupter filipin (Figure 6C), suggesting a higher order of lipid rafts in Cd82-null EC membrane. Consistently, the surface levels of ganglioside GM1, GM2, and GM3 were upregulated in Cd82-null ECs (Figure 6D). Ceramide and cholesterol levels remained unaltered.
Detergent solubility of CAMs and tetraspanin was reduced on Cd82 ablation even after cytoskeletal disruption (Figure XIII in the online-only Data Supplement), suggesting that CD82 mainly modulates membrane compartmentalization of these proteins (see the Results section in the online-only Data Supplement). Because in TEMs CD82 associates with tetraspanins and CAMs,9 we analyzed TEMs through immunoprecipitation profiles (Figure 6E). CD9 and CD9-associated unknown surface proteins (X1 and X2), not CD9-associated integrins and CD81, were increased in Cd82-null ECs, suggesting that the extra CD9 proteins at the EC surface are integrin and CD81 free. Levels and species of the surface proteins associated with β1 integrins and CD44 were generally unchanged on Cd82 ablation, despite CD44 upregulation. Thus, integrin-CD9 association and CD44 complex, not CD9-containing TEM, remain unaltered without CD82.
To confirm the roles of lipid raft clustering and ganglioside increase in angiogenesis, we examined the effects of filipin and PDMP, a glucosylceramide synthase inhibitor that blocks ganglioside formation, on aortic ring angiogenesis and EC motility. Filipin and PDMP diminished the difference in angiogenesis between the WT and KO groups and reduced Cd82-null EC cell motility to the WT level (Figure 6F and 6G).
CD82-Ganglioside-CD44 Signaling and Pathological Angiogenesis
Increased angiogenic potential in Cd82-null mice makes us question whether CD82 is downregulated during pathological neovascularization. Indeed, vascular expression of CD82 was largely diminished in proliferative diabetic retinas, which is caused by profound angiogenesis, compared with CD82 expression in the vessels of normal retina (Figure XIV in the online-only Data Supplement). Moreover, we compared vascular expressions of CD82 in human normal breast and breast cancer tissues using tissue microarray and found that CD82 proteins in the blood vessels within invasive breast cancer were reduced compared with those in normal breast tissue (Figure 7A, arrowheads). However, vascular CD44 exhibited a converse pattern, indicating that reduced CD82 and enhanced CD44 coexist in human tumor angiogenesis. The same changes in CD82 and CD44 expression were also observed from epithelial to tumor cells.
Our study reveals that CD82-ganglioside-CD44 signaling is connected to angiogenic potential (Figure 7B). We perturbed this signaling in vivo to confirm its importance. With administration of PDMP or CD44 shRNA in Matrigel plug angiogenesis assay, the increased angiogenesis in Cd82-null group was reduced to the levels of WT group (Figure 7B), indicating that increased gangliosides and CD44 are essential for the enhanced angiogenesis in vivo on Cd82 ablation.
Here, we identified the confinements of EC movement and angiogenic potential as novel functions for CD82, demonstrated that lipid raft clustering and CAM trafficking modulate angiogenic potential, and revealed a novel mechanistic paradigm that membrane glycosphingolipids tune angiogenic potential through altering CAMs at the plasma membrane. Because no gross vascular abnormality was observed after development, CD82 likely restrains EC movement to suppress angiogenesis under pathological conditions and prevents excessive vascular morphogenesis under physiological conditions. In addition, our study conceptualizes the schemes that membrane protein regulates lipid rafts and that membrane compartmentalization of CAMs modulates EC movement. To inhibit EC movement, CD82 likely enhances CAM endocytosis by changing CAM-membrane microdomain interactions. Without CD82, higher magnitudes of CAMs and CAM-initiated signaling at the EC surface more efficiently induce focal adhesive structures and microextrusions, drive EC movement, and subsequently facilitate pathological angiogenesis (Figure XV in the online-only Data Supplement).
CD82 Confines Angiogenesis Mainly by Restraining EC Movement
Although CD82 is expressed in ECs27 and Cd82-null mice exhibit elevated vascular morphogenic potential, these mice do not display obvious vascular defects, suggesting that CD82 is not essential for physiological vessel development. However, it is unlikely to have developmental vascular defects when vascular morphogenic potential is above normal. In addition, increased vascular morphogenesis is not needed for normal development of animals. Increased pathological angiogenesis in Cd82-null mice underlines that loss of CD82 function cannot be mitigated or compensated for after development and that CD82 inhibits molecular and cellular events unique to or critical for pathological angiogenesis, which is distinct from physiological angiogenesis.28 Our observations highlight that pathological angiogenesis lacks an efficient regulatory mechanism for EC movement, which is the main cellular event that CD82 controls during angiogenesis, and suggest that downregulating EC movement serves a therapeutic strategy selectively against pathological angiogenesis.
Pathological angiogenesis is characterized by its morphogenic simplicity.29 In Cd82-null mice, the increased angiogenic potential is associated with more rapid and efficient formation of capillaries, driven mainly by enhanced EC movement. In other words, the relative simplistic morphogenic program of pathological angiogenesis depends more on EC movement than significantly more complex physiological angiogenesis does. Notably, angiogenesis without CD82 is functional, as evidenced by better blood-perfused Matrigel plug, larger tumor, more perfused vascular tufts, and stronger cardiac function after MI in Cd82-null mice.
CD82 Restrains EC Movement by Inhibiting CAMs
CD82-dependent alteration in cell adhesion is likely to be directly responsible for the change in movement. For example, enhanced adhesion onto hyaluronan promotes EC infiltration in interstitial tissue, whereas upregulated FAK/Src-p130CAS and Akt activities serve as promigratory signaling in Cd82-null ECs.
Tetraspanins preferentially associate with laminin-binding integrins like α6 integrins. The upregulated α6 integrins correlate with the enhanced Cd82-null EC adhesion onto laminin 111. Interestingly, the upregulated αV integrins and unchanged α5β1 integrin, both RGD-binding integrins, did not enhance Cd82-null EC adhesion onto fibronectin. Hence, whether CD82 affects integrin activation remains to be determined. Alternatively, the upregulated α6 and αV integrins may contribute to other activities such as Notch and Netrin signaling.30,31 To visualize how CD82, CD44, and integrin are related at the molecular level, we cross-referenced the top 20 genes most frequently cotranscribed with CD82 with their known protein-protein interactions (Figure XVI in the online-only Data Supplement). Most of the 4 known protein-protein interactions shared by CD82 and CD44 are associated with cell migration and angiogenesis, suggesting potential genetic partners by which the 2 may exert their phenotype-altering influence.
CD44 and tetraspanins activate multiple signaling pathways.8,32 FAK/Src-p130CAS and Akt signaling affects cell adhesion and movement and is altered on CD82 overexpression.8,9 CD44 partitions to lipid rafts and associates with Src through its cytoplasmic domain.33,34 The increased FAK/Src-p130CAS activity in Cd82-null ECs likely results from the elevated levels or altered microdomain coalescences of CAMs like CD44 at the plasma membrane. Higher Akt activity could also be caused by more surface CAMs because hyaluronan-CD44 binding activates PI-3 kinase–Akt signaling.32 Src is likely situated between CD44 and Akt because Src indirectly activates PI-3 kinase and Akt during angiogenesis,35 and Src maximally activates FAK through FAK-Y577 phosphorylation. We propose that CD82 transdominantly inhibits CD44 and its downstream signaling by modulating the membrane microdomain coalescence of CD44 to confine EC-hyaluronan adhesion. Consistently, CD44 has proangiogenic properties, evidenced by the observations that vascular morphogenesis becomes attenuated in Cd44-null mice, CD44 antibodies inhibit EC proliferation and vascular morphogenesis, and CD44v6 serves as a coreceptor for c-Met and vascular endothelial growth factor receptor-2 in ECs during angiogenesis.19,22,36–38 Our study further revealed how CD44 promotes angiogenesis at the molecular level.
CD82 Inhibits CAMs by Altering the Microdomain Coalescence and Then Endocytosis of CAMs
CD44 perturbation reduced the enhanced migration and angiogenesis of the Cd82-null group to the levels of WT, supporting the notion that CD44 has immediate and major functional connections to CD82. Reductions were also found in the WT group, suggesting that CD44 also controls these events at physiological conditions.
The increased expression of CD44, integrin α6, CD9, and gangliosides at Cd82-null EC surface likely results from their decreased turnover at the plasma membrane. Less endocytosis of CD44 apparently causes more CD44 at the Cd82-null EC surface. Because cholesterol is important for clathrin-independent/CLIC endocytic pathway,39,40 CD82 modulates this pathway probably by reorganizing saturated lipids into or between membrane microdomains.12 Increased gangliosides in Cd82-null EC plasma membrane likely facilitate lipid raft clustering in a cholesterol-dependent manner, as found in the model membrane,41 and coalescence of CD44 to TEMs and lipid rafts because tetraspanins physically interact with CD44 and gangliosides.11,42 We predict that microdomain reorganization leads to less endocytosis of microdomain residents such as CD44, CD9, and gangliosides.
Stronger cell-matrix adhesiveness and greater focal adhesive structures in Cd82-null ECs reflect a robust membrane-cytoskeleton connection, which may reduce CAM endocytosis. However, increased interactions of CD44 with lipid rafts and TEMs likely play more dominant roles in its trafficking because CD44 is internalized through a clathrin-independent, raft-dependent pathway.23,24
CD82 Is a Lipid Raft Organizer
Robust microextrusions in Cd82-null ECs likely result from the reorganized membrane microdomains and contribute to active motile behaviors and strong adhesiveness during angiogenesis, given that microextrusions may modulate cell adhesion and movement.18
Tetraspanins had not been found in focal complex and adhesion,8 the membrane microdomains of cytoskeleton-connected CAM clusters. Upon Cd82 ablation, redistribution of tetraspanins/TEMs to focal complexes further corroborates CD82 as a membrane domain organizer. Because TEMs contain various CAMs, such redistribution suggests greater clustering of CAMs and stronger EC adhesion strengthening.
CD82-Ganglioside-CD44 Signaling in Angiogenesis
Ganglioside buildup on Cd82 removal and subsequent lipid raft clustering upregulate CD44 in pathological angiogenesis, which can be attenuated by inhibiting CD82-ganglioside-CD44 signaling. Although filipin could exert a broader effect on cells, the notion that filipin inhibits angiogenesis by disrupting lipid rafts is supported by the effects of ganglioside reduction and CD44 blockade.
Our study revealed that the membrane microdomain landscape plays a key role in pathological angiogenesis and delineated that CD82 modulates CAM trafficking and then surface expression by altering lipid rafts clustering (Figure XIV in the online-only Data Supplement). Importantly, we first demonstrated that CD82 protein drives lipid raft reorganization. Because angiogenesis is linked to many diseases, our observations have far-reaching implications. Future studies will determine how CD82 alters membrane lipids and evaluate the therapeutic potentials of CD82-ganglioside-CD44 signaling against pathological angiogenesis. Studies on CD82 and tumor progression have so far focused on the metastasis-suppressive effect that CD82 exerts directly on tumor cells.9 Given that endothelial CD82 inhibits tumor angiogenesis, CD82 can be a drug candidate with dual benefits against tumor progression.
Dr Zhang is an Oklahoma TSET Cancer Research Scholar. We thank Drs S. Vesely, Y. Chen, S. Frank, R. McEver, L. Xia, H. Chen, C. Griffin, and K. Kyler for discussions; Dr X. Qi for technical support; and the OMRF imaging facility for confocal microscopy.
Sources of Funding
This study was funded by National Institutes of Health grant CA096991 and American Heart Association grant 13GRNT17040028 to Dr Zhang.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.114.011096/-/DC1.
- Received January 21, 2014.
- Accepted August 18, 2014.
- © 2014 American Heart Association, Inc.
- Hanahan D
- Davis GE,
- Senger DR
- Odintsova E,
- Butters TD,
- Monti E,
- Sprong H,
- van Meer G,
- Berditchevski F
- Todeschini AR,
- Dos Santos JN,
- Handa K,
- Hakomori SI
- Xu C,
- Zhang YH,
- Thangavel M,
- Richardson MM,
- Liu L,
- Zhou B,
- Zheng Y,
- Ostrom RS,
- Zhang XA
- Roy NH,
- Chan J,
- Lambelé M,
- Thali M
- Schwenk F,
- Baron U,
- Rajewsky K
- Golshani R,
- Lopez L,
- Estrella V,
- Kramer M,
- Iida N,
- Lokeshwar VB
- Savani RC,
- Cao G,
- Pooler PM,
- Zaman A,
- Zhou Z,
- DeLisser HM
- Tremmel M,
- Matzke A,
- Albrecht I,
- Laib AM,
- Olaku V,
- Ballmer-Hofer K,
- Christofori G,
- Héroult M,
- Augustin HG,
- Ponta H,
- Orian-Rousseau V
- Howes MT,
- Kirkham M,
- Riches J,
- Cortese K,
- Walser PJ,
- Simpson F,
- Hill MM,
- Jones A,
- Lundmark R,
- Lindsay MR,
- Hernandez-Deviez DJ,
- Hadzic G,
- McCluskey A,
- Bashir R,
- Liu L,
- Pilch P,
- McMahon H,
- Robinson PJ,
- Hancock JF,
- Mayor S,
- Parton RG
- Tammi R,
- Rilla K,
- Pienimaki JP,
- MacCallum DK,
- Hogg M,
- Luukkonen M,
- Hascall VC,
- Tammi M
- Chichili GR,
- Cail RC,
- Rodgers W
- Estrach S,
- Cailleteau L,
- Franco CA,
- Gerhardt H,
- Stefani C,
- Lemichez E,
- Gagnoux-Palacios L,
- Meneguzzi G,
- Mettouchi A
- Larrieu-Lahargue F,
- Welm AL,
- Thomas KR,
- Li DY
- Bourguignon LY,
- Zhu H,
- Shao L,
- Chen YW
- Oliferenko S,
- Paiha K,
- Harder T,
- Gerke V,
- Schwärzler C,
- Schwarz H,
- Beug H,
- Günthert U,
- Huber LA
- Kumar P,
- Amin MA,
- Harlow LA,
- Polverini PJ,
- Koch AE
- Griffioen AW,
- Coenen MJ,
- Damen CA,
- Hellwig SM,
- van Weering DH,
- Vooys W,
- Blijham GH,
- Groenewegen G
- Yu Q,
- Stamenkovic I
- Damm EM,
- Pelkmans L,
- Kartenbeck J,
- Mezzacasa A,
- Kurzchalia T,
- Helenius A
- Lingwood D,
- Ries J,
- Schwille P,
- Simons K
- Mitsuzuka K,
- Handa K,
- Satoh M,
- Arai Y,
- Hakomori S
Angiogenesis is fundamentally important for the pathogenesis and progression of various diseases, including cardiovascular diseases such as myocardial infarction and stroke. Selective, efficient, and persistent perturbation of angiogenesis for the purposes of disease treatment is still beyond our reach. The limited efficacy of the angiogenesis therapy based on vascular endothelial growth factor and fibroblast growth factor antagonism highlights that the mechanism of pathological angiogenesis is unique. In addition to unveiling the inhibitory roles of tetraspanin CD82 in endothelial cell movement and pathological angiogenesis, our study reveals a novel angiogenesis-regulatory mechanism by which membrane glycosphingolipids and their derived lipid rafts in endothelial cells modulate angiogenic potential. Our study also presents CD82-ganglioside-CD44 signaling as a potential therapeutic target against angiogenesis. More important, our findings provide a novel strategy to intervene angiogenesis under pathological conditions, that is, the reorganization of membrane microdomains at the endothelial cell surface. Together with earlier observations of tetraspanin CD151, this study of tetraspanin CD82 supports an emerging notion that tetraspanins could be clinically beneficial through upregulation or downregulation of vascular functions such as endothelial cell movement and angiogenesis.