TY - JOUR
AU - Wu,, Yi
AB - Abstract Aims Circulating proteins larger than 3 nm can be transported across continuous endothelial barrier of blood vessels via transcytosis. However, excessive accumulation of serum proteins within the vessel walls is uncommon even for those abundant in the circulation. The aim of this study was to investigate how transcytosis regulates tissue accumulation of the prototypical acute-phase reactant C-reactive protein (CRP) and other serum proteins. Methods and results Transcytosis of CRP as well as of transferrin and low-density lipoprotein across aortic endothelial cells is bidirectional with directional preference from the apical (blood) to basolateral (tissue) direction both in vitro and in vivo. This directional preference is, however, reversed by the basement membrane (BM) matrix underlying the basolateral surface of endothelial cells. This is due to the sieving effect of the BM that physically hinders the diffusion of transcytosed proteins from the apical compartment towards underlying tissues, resulting in immediate retrograde transcytosis that limits basolateral protein accumulation. Conversely, CRP produced within vessel wall lesions can also be transported into the circulation. Conclusion Our findings identify matrix sieving-enforced retrograde transcytosis as a general mechanism that prevents excessive tissue accumulation of blood-borne proteins and suggest that lesion-derived CRP might also contribute to elevated serum CRP levels associated with increased risk for cardiovascular diseases. Inflammation , Atherosclerosis , Transcytosis , Basement membrane, C-reactive protein 1. Introduction The luminal surface of most blood vessels including arteries, is lined by a continuous monolayer of endothelial cells, which forms a selective barrier to the exchange of macromolecules between the circulation and surrounding tissues.1 Transcytosis is the major route for circulating proteins larger than 3 nm to cross the endothelial barrier.2,3 C-reactive protein (CRP) is a well-established circulating marker of inflammation. CRP is predominantly produced by the liver and has a diameter over 10 nm.4,5 Blood concentrations of CRP can increase from less than 1 μg/mL to over 500 μg/mL within 24 h in response to infection or tissue injury. Despite its abundance in the circulation, blood vessels from apparently healthy subjects stain negative for CRP,6–8 and 125I-CRP injected intravenously into patients with different focal pathologies does not accumulate in tissue lesions.9 These findings would suggest that circulating CRP does not undergo transcytosis, hence cannot be transported into tissues across an intact endothelial barrier. An important implication of this notion is that CRP produced by the liver may function predominantly in the circulation.9 Conversely, CRP detected within tissue lesions, including initial and advanced atherosclerotic plaques6–8 in the aorta and coronary arteries with intact endothelial barrier, might have been produced in situ and would act locally. In this study, we investigated whether CRP can undergo transcytosis both in vitro and in vivo. We found that CRP readily underwent transcytosis from the apical (blood) to the basolateral compartment of the endothelium (tissue). However, the sieving effect of the basement membrane (BM) beneath endothelial cells enforced an immediate retrograde transportation, thereby limiting tissue accumulation of CRP and other serum proteins. Moreover, CRP produced within the vessel wall can also be transported from the basolateral to the apical compartment. These findings uncover a novel physical mechanism by which the BM modulates the directionality of transcytosis and consequently contributes to maintaining tissue homeostasis. 2. Material and methods 2.1 Reagents Human native CRP (purity > 99%) purified from ascites was purchased from the BindingSite (Birmingham, UK; catalog number: BP300.X). Proteins were repurified with phosphorylcholine (PC)-agarose beads, dialyzed to remove NaN3, and passed through Detoxi-Gel Columns (Thermo Fisher Scientific, Rockford, IL; catalog number: 20344) to remove endotoxin when necessary. CRP was labelled with FITC (Sigma-Aldrich, St. Louis, MO; catalog number: F4274) or Alexa Fluor 594 Protein Labeling Kit (Thermo Fisher Scientific; catalog number: A10239) according to the manufacturer’s instruction. Transferrin was purchased from the Solarbio (Beijing, China; catalog number: 11096-37-0; lot number: 713C047) and labelled with FITC (Sigma-Aldrich; catalog number: F4274). DiI-labeled LDL was purchased from Yeasen (Shanghai, China; catalog number: 20606ES76; lot number: H77310). Mouse anti-human CRP mAbs were generated as described.10 2.2 Transwell culture of endothelial cells Primary human aortic endothelial cells (HAEC; Lifeline Cell Technology, Frederick, MD; catalog number: FC-0027; lot number: 01381; PromoCell, Heidelberg, Germany; catalogue number: C-12271; lot number: 4082102.16; Cascade Biologics, Portland, OR; catalog number: C-006-5C; lot number: 5C0619, 5C1143, and 765093), human umbilical vein endothelial cells (HUVEC; Cascade Biologics; catalog number: C-003-5C; lot number: 4C0218), and human coronary artery endothelial cells (HCAEC, Lifeline Cell Technology; catalog number: FC-0032; lot number: 01181; Cell Applications, SanDiego, CA; catalog number: 300-05a; lot number: 2463, 2827, and 2139) were cultured as described.8,11 A 105 ECs at passages 4–6 were seeded on filters of Transwell coated with 36 μg rat tail collagen I (Corning, NY; catalog number: 354236; lot number: 5036302) for 8 h followed by washing to remove non-adherent cells.12 In some experiments, filters were first coated with 120 μL pre-thawed Matrigel (Corning, catalog number: 354234; lot number: 6088006) at 4°C. The filters were next incubated at 37°C for 30 min to form well-organized ECM layer. Rat tail collagen I was then applied on top of the Matrigel layer followed by seeding of ECs. The culture media were changed every 2 days and the formation of intact EC monolayers was assessed by crystal violet staining, fluorescence imaging, and the transendothelial electrical resistance measured by Millicell-ERS (Millipore, Billerica, MA, USA). 2.3 Transcytosis assay Confluent EC monolayers on Transwell filters were gently washed with HBSS. CRP, transferrin or LDL at the indicated concentrations were added to the apical or basolateral chambers of Transwell at 37°C for the indicated times. Media in the opposite chambers were collected and the transcytosis was quantified by ELISA or fluorescence intensity. Preference index was calculated as (the amount of CRP transcytosed in the apical-to-basolateral direction)/(the amount of CRP transcytosed in the basolateral-to-apical direction). For receptor overexpression, FcγR1A, FcγR2B, or LOX-1 was cloned into pWSLV-05 vector and transfected into HAECs (Lifeline Cell Technology; catalog number: FC-0027; lot number: 01381) using lentivirus particles at a MOI of 40 produced by HEK293T cells (National Infrastructure of Cell Line Resources, Shanghai, China; catalog number: 3111C0001CCC000010). Cells were then seeded onto Transwell filters for transcytosis assay. For receptor blockade, 10 μg/mL anti-FcγR1 (Santa Cruz, Dallas, TX; catalog number: sc-1184; lot number: K0409), anti-FcγR2 (Santa Cruz; catalog number: sc-13527; lot number: GR254149-5), or anti-LOX1 (Abcam, Cambridge, UK; catalog number: ab60178; lot number: GR225261-13) was added to both the apical and basolateral chambers of Transwell with HAEC monolayers for 15 min before addition of CRP and were present during the transcytosis assay. 2.4 Fluorescence imaging To visualize the transcytosis of CRP, 20 μg/mL FITC-labeled CRP was added to apical chambers of Transwell with confluent HAEC monolayers at 37°C for 40 min. Cells were fixed with 4% paraformaldehyde for 30 min and incubated with 5 μg/mL anti-VE-cadherin mAb BV9 (Santa Cruz; catalog number: sc-52751; lot number: J0914) at 4°C for 1 h. After extensive washing, Alexa-647 goat anti-mouse IgG H&L (1:300; Abcam; catalog number: ab150115; lot number: GR309891-3) was added at 4°C for 1 h. Nuclei were counterstained with DAPI (Southern Biotech, Birmingham, AL; catalog number: 0100-20; lot number: F0617-S327) for 15 min at room temperature. To visualize the interaction of CRP with its receptors, HAEC monolayers (Lifeline Cell Technology; catalog number: FC-0027; lot number: 01381) cultured on Transwell filters were transfected with mCherry-tagged FcγR1A, FcγR2B or LOX-1 in pmCherry-N1 vector (Clontech, Mountain View, CA; catalog number: 632523) using lipofectamine 3000 reagents (Invitrogen, Carlsbad, CA; catalog number: L3000-015; lot number: 1811507). A total of 20 μg/mL FITC-labeled CRP was added to the apical chambers for 15 min at 4°C followed by incubation at room temperature for 30 min. Cells were fixed and counterstained with DAPI. To analyse the intracellular route of CRP, HAEC monolayers were incubated with 20 μg/mL FITC-labeled CRP for 60 min at 37°C. Cells were fixed with 4% paraformaldehyde for 30 min followed by permeabilization with 0.4% Triton X-100 for 10 min at 4°C. After blocking with 5% BSA, early endosomes were marked with an anti-EEA1 antibody (1:200; BD Biosciences, San Jose, CA; catalog number: 610457; lot number: 12423) and Alexa-647 goat anti-mouse IgG H&L (1:300; Abcam; catalog number: ab150115; lot number: GR309891-3), Golgi apparatus were marked with an anti-TGN46 antibody (1 μg/mL; Abcam; catalog number: ab76282; lot number: GR112320-5) and Dylight-550 goat anti-rabbit IgG H&L (1:500; Abcam; catalog number: ab96884; lot number: GR162633-3), recycling endosomes were marked with an anti-Rab11a antibody (1 μg/mL; Abcam, Cambridge, UK; catalog number: ab65200; lot number: GR150456-1) and Dylight-550 goat anti-rabbit IgG H&L (1:500; Abcam; catalog number: ab96884; lot number: GR162633-3), lysosomes were marked by expression of mCherry-Lamp1, and microtubules were marked with an anti-α-Tubulin mAb (1:400; EarthOx Life Sciences, Millbrae, CA; catalog number: E021030-01; lot number: 70003) and Alexa-647 goat anti-mouse IgG H&L (1:400; Abcam; catalog number: ab150115; lot number: GR309891-3). Samples were examined by a LSM 710 confocal microscopy (Zeiss, Jena, Germany). Technical controls without addition of labelled CRP or primary antibodies showed negligible background signals. 2.5 Total internal reflection fluorescence imaging HAECs cultured on ultra-thin cover glasses in 35-mm glass bottom dishes were incubated with 50 µg/mL Alexa 568-labeled CRP at 4°C for 25 min, rinsed with cold PBS, and then incubated at 37°C for 5 min. Samples were excited with lasers at 561 nm, and total internal reflection fluorescence (TIRF) images were acquired with an ECLIPSE Ti (Natori, Japan) equipped with an iXon DU-897E EMCCD camera (Andor, Belfast, UK) using ×100 objective lens (NA = 1.49) immersed in oil. The penetration depth was at 110 nm. Images were analysed with Imaris 8 (Bitplane, Zurich, Switzerland) and Image J packages (NIH). Following correction for noise and local background, vesicles were identified according to size (0.15∼0.65 μm) and sphericity (over 0.2). Tracks of vesicles were determined with autoregressive motion tracking algorithm with quality control parameters including Tack Displacement Length (0.00–1.00 μm), Track Speed Variation (0–1), Track Intensity StdDev (over 35), and Track Duration (1∼60 s). Our analysis was therefore focused on docked vesicles that would undergo exocytosis or endocytosis. Exocytotic events were identified as rapid loss of signal,13 while endocytic events were quite slower.14 2.6 Tissue infiltration of circulating CRP Non-labelled or Alexa 568-labeled human CRP was injected intravenous into male Kunming mice (25 ± 2 g; housed under specific pathogen-free conditions) at a dose of 2.5 mg/kg. To assess tissue accumulation of non-labelled CRP, mice were anaesthetized with Isoflurane (3% for induction and 1.5% thereafter) and aortas were collected following perfusion. Tissues were lysed with RIPA buffer (Solarbio; catalog number: R0020; lot number: 20160406) containing PMSF. CRP accumulated in the aorta was semi-quantified with immunoblotting, while serum CRP levels were determined with ELISA.12 To visualize tissue distribution of labelled CRP, 8-μm thick cryosections were prepared, fixed in cold acetone for 15 min, washed with PBS and then blocked with 1% BSA and 3% FBS for 1 h at room temperature. The sections were incubated with an anti-Laminin antibody (1:500; Abcam; catalog number: ab11575; lot number: GR308278-1) at 4°C overnight followed by Alexa-647 goat anti-mouse IgG H&L (1:300; Abcam; catalog number: ab150115; lot number: GR309891-3) for 1 h at room temperature and DAPI counterstaining. The sections were then examined by a LSM 710 confocal microscopy. The animal experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the protocols were approved by the Ethics Committee of Animal Experiments of Xi'an Jiaotong University (permit number: 2016-064). 2.7 Tissue contribution to circulating CRP Alexa 568-labeled human CRP was injected intravenously into male Kunming mice (25 ± 2 g) at a dose of 2.5 mg/kg for 40 min. Mice were then perfused with PBS for 90 min, selected organs were collected, cryosectioned, stained, and visualized. In another set of experiments, CT26 colon carcinoma cells stably expressing a control vector or human CRP (coding sequence plus signal peptide) were inoculated s.c. into male BALB/c mice at 8 weeks of age. Serial venous blood samples were collected, and levels of human CRP were detected with a specific ELISA.12 2.8 Statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed by two-tailed Student’s t-test, one-way ANOVA with Tukey post hoc or Kolmogorov–Smirnov test or Mann–Whitney’s U-test as appropriate. Differences were considered significant at values of P < 0.05. 3. Results 3.1 CRP can be transcytosed across intact HAEC monolayers We first examined whether CRP can undergo transcytosis in a Transwell model, in which confluent monolayers of primary HAECs were grown on porous filters with their apical and basolateral surfaces exposed to separate chambers (Figure 1A). Addition of CRP to the apical chamber that mimics blood vessel lumen was associated with increases in CRP level in the basolateral chamber (that corresponds tissue) over time (Figure 1B). Lowering temperature markedly inhibited these increases (Figure 1B). The basolateral accumulation of apically added CRP were over 20-fold higher across blank filters vs. filters covered with HAEC monolayers (Figure 1C). Confocal imaging confirmed that apically applied CRP was internalized as puncta and penetrated the entire thickness of the HAEC monolayer (Figure 1D). Moreover, TIRF microscopy revealed that the internalized CRP was exocytosed at the basolateral surface of HAEC (Figure 1E). Taken together these results identify an active, vesicle-mediated trans-endothelial transportation of CRP, i.e. transcytosis. Figure 1 View largeDownload slide CRP undergoes transcytosis across intact HAEC monolayers. (A) The experimental setup for investigating CRP transcytosis across HAEC monolayers cultured on Transwell filters (pore size of 0.4 μm) (left). The formation of continuous HAEC monolayers on Transwell filters was confirmed by crystal violet staining (middle) and fluorescent imaging of VE-cadherin (right; red-coloured) (n = 5). (B) CRP (20 μg/mL) was added to the apical chambers and transcytosis was assayed at 37°C and 16°C (n = 5). CRP concentrations in the basolateral chambers were determined with ELISA. (C) CRP transportation across Transwell filters without HAEC monolayers (n = 5). (D) CRP internalization and transport to basolateral surfaces. Alexa-568-labeled CRP (20 μg/mL) was added to the apical chambers of Transwell with intact HAEC monolayers for 40 min. X–Y and X–Z images of one representative cell (left), and the averaged intensity distribution along Z axis (right) are shown (n = 8). (E) Alexa-568-labeled CRP (50 μg/mL) was added to the apical surfaces of HAEC monolayers cultured on ultra-thin cover slips at 4°C for 25 min. Following extensive washing, cells were incubated at 37°C for 5 min to internalize surface-bound CRP, and the basolateral surfaces were observed with TIRF (left) (n = 9). Exocytosis of CRP-containing vesicles were noted at the basolateral surfaces. Three representative events of exocytosis (middle) and their intensity-time profiles (right) are shown. Values are mean ± SEM, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). Figure 1 View largeDownload slide CRP undergoes transcytosis across intact HAEC monolayers. (A) The experimental setup for investigating CRP transcytosis across HAEC monolayers cultured on Transwell filters (pore size of 0.4 μm) (left). The formation of continuous HAEC monolayers on Transwell filters was confirmed by crystal violet staining (middle) and fluorescent imaging of VE-cadherin (right; red-coloured) (n = 5). (B) CRP (20 μg/mL) was added to the apical chambers and transcytosis was assayed at 37°C and 16°C (n = 5). CRP concentrations in the basolateral chambers were determined with ELISA. (C) CRP transportation across Transwell filters without HAEC monolayers (n = 5). (D) CRP internalization and transport to basolateral surfaces. Alexa-568-labeled CRP (20 μg/mL) was added to the apical chambers of Transwell with intact HAEC monolayers for 40 min. X–Y and X–Z images of one representative cell (left), and the averaged intensity distribution along Z axis (right) are shown (n = 8). (E) Alexa-568-labeled CRP (50 μg/mL) was added to the apical surfaces of HAEC monolayers cultured on ultra-thin cover slips at 4°C for 25 min. Following extensive washing, cells were incubated at 37°C for 5 min to internalize surface-bound CRP, and the basolateral surfaces were observed with TIRF (left) (n = 9). Exocytosis of CRP-containing vesicles were noted at the basolateral surfaces. Three representative events of exocytosis (middle) and their intensity-time profiles (right) are shown. Values are mean ± SEM, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). We next sought to identify the receptor that mediates the transcytosis of CRP. Of the three known receptors for CRP on ECs,15–17 FcγR1A, and FcγR2B but not LOX-1 appeared to be bound and internalized with CRP (Figure 2A). Although CRP was added apically, its colocalization with the two FcγRs extended from the apical to the basolateral surfaces, suggesting the involvement of both receptor types in CRP transcytosis. Indeed, overexpression or blockade of FcγR1A enhanced or reduced CRP transcytosis, respectively (Figure 2B–E). In contrast, overexpression or blockade of FcγR2B was associated with modest changes in CRP transcytosis, whereas altering LOX-1 expression or function had no detectable effects (Figure 2B–E). Additional experiments revealed that CRP was internalized via clathrin-dependent endocytosis (Figure 2F). Internalized CRP was co-localized with early endosomes but barely with recycling endosomes, lysosomes or trans-Golgi apparatus (Figure 2G). Most CRP-positive vesicles were also well aligned with microtubules (Figure 2H), the major tracks for long-range cellular transportation.3 Therefore, CRP uptaken by clathrin-coated pits are unlikely to be destined for degradation; instead, they are sorted to the pathway of transcytosis mediated partly by FcγR. Figure 2 View largeDownload slide FcγR1A mediates predominantly the transcytosis of CRP. (A) FITC-labeled CRP (20 μg/mL) was added to the apical chamber of Transwell with intact HAEC monolayers transfected with mCherry-tagged FcγR1A, FcγR2B or LOX-1. Confocal imaging shows extensive colocalization of CRP with FcγR1A and FcγR2B spanning the whole thickness of HAEC monolayers. Representative X–Y and X–Z sections are shown (n = 5–8). (B) Transcytosis of apically added CRP across HACE monolayers was significantly enhanced by overexpression of FcγR1A, but not by overexpression of FcγR2B or LOX-1 (n = 3). (C) Effects of receptor blockade. FITC-labeled CRP (20 μg/mL) was added to the apical chamber of Transwell with intact HAEC monolayers in the absence or presence of 10 μg/mL blocking antibodies against FcγR1A, FcγR2B, or LOX-1. An isotope-matched irrelevant antibody was without effects. Representative X–Y and X–Z sections of confocal imaging are shown. (D) Quantitative analysis of confocal imaging. The boxes indicate the interquartile range between the 25th and 75th percentiles (n = 6–8). (E) Effects of FcγR1A, FcγR2B, or LOX-1 blockade on transcytosis of apically added CRP (n = 5). (F) Effects of endocytosis inhibitors. FITC-labeled CRP (50 μg/mL) was added to the apical side of HAEC monolayer in the absence or presence of the inhibitors of clathrin-dependent endocytosis Pitstop (30 μM, 15 min prior to CRP) or Dynasore (80 μM, 30 min prior to CRP), or MβCD (0.5 mM, 15 min prior to CRP), which inhibits cholesterol-dependent endocytosis (n = 6). (G, H) Colocalization of CRP with intracellular organelles and alignment with microtubules. FITC-labeled CRP (20 μg/mL) was added to the apical side of HAEC monolayer for 45 min, and its colocalization with EEA1, Rab11a, LAMP1, TNG46 (n = 3–5) (G) and microtubules (n = 5) (H) was visualized with confocal microscopy. Representative images are shown. Values are mean ± SEM, *P < 0.05, **P < 0.01 (Kolmogorov–Smirnov test for D; two-tailed Student’s t-test for F). Figure 2 View largeDownload slide FcγR1A mediates predominantly the transcytosis of CRP. (A) FITC-labeled CRP (20 μg/mL) was added to the apical chamber of Transwell with intact HAEC monolayers transfected with mCherry-tagged FcγR1A, FcγR2B or LOX-1. Confocal imaging shows extensive colocalization of CRP with FcγR1A and FcγR2B spanning the whole thickness of HAEC monolayers. Representative X–Y and X–Z sections are shown (n = 5–8). (B) Transcytosis of apically added CRP across HACE monolayers was significantly enhanced by overexpression of FcγR1A, but not by overexpression of FcγR2B or LOX-1 (n = 3). (C) Effects of receptor blockade. FITC-labeled CRP (20 μg/mL) was added to the apical chamber of Transwell with intact HAEC monolayers in the absence or presence of 10 μg/mL blocking antibodies against FcγR1A, FcγR2B, or LOX-1. An isotope-matched irrelevant antibody was without effects. Representative X–Y and X–Z sections of confocal imaging are shown. (D) Quantitative analysis of confocal imaging. The boxes indicate the interquartile range between the 25th and 75th percentiles (n = 6–8). (E) Effects of FcγR1A, FcγR2B, or LOX-1 blockade on transcytosis of apically added CRP (n = 5). (F) Effects of endocytosis inhibitors. FITC-labeled CRP (50 μg/mL) was added to the apical side of HAEC monolayer in the absence or presence of the inhibitors of clathrin-dependent endocytosis Pitstop (30 μM, 15 min prior to CRP) or Dynasore (80 μM, 30 min prior to CRP), or MβCD (0.5 mM, 15 min prior to CRP), which inhibits cholesterol-dependent endocytosis (n = 6). (G, H) Colocalization of CRP with intracellular organelles and alignment with microtubules. FITC-labeled CRP (20 μg/mL) was added to the apical side of HAEC monolayer for 45 min, and its colocalization with EEA1, Rab11a, LAMP1, TNG46 (n = 3–5) (G) and microtubules (n = 5) (H) was visualized with confocal microscopy. Representative images are shown. Values are mean ± SEM, *P < 0.05, **P < 0.01 (Kolmogorov–Smirnov test for D; two-tailed Student’s t-test for F). 3.2 CRP transcytosis is polarized to apical-to-basolateral direction The ability of HAECs to transcytose CRP would suggest that this molecule could be a substrate for blood-tissue exchange. To determine whether the exchange shows directional preference for blood or tissue CRP, we compared CRP transcytosis by HAECs in the apical-to-basolateral (i.e. blood-to-tissue) and the reverse direction (Figure 3A). We found that the apical-to-basolateral transcytosis was at least two-fold stronger than in the opposite direction over the entire time course studied (Figure 3B). This directional preference was consistently observed at various CRP concentrations tested (Figure 3B), using filters with distinct specifications (Figure 3C) as well as with different types of EC (Figure 3D). Pathological stimuli, such as IFN-γ or hyperglycaemia enhanced CRP transcytosis in both directions without eliminating the directional preference (Figure 3E). The directional preference cannot be attributed to the small difference between apical and basolateral accessibility, for up to 80% of the filter area is decorated by pores. Moreover, Lucifer yellow, a tracer of paracellular transport, showed no directional preference in transportation across HAECs (Figure 3F). These results together would imply a blood-to-tissue dominated exchange of CRP. Figure 3 View largeDownload slide The bidirectional transcytosis of CRP prefers to apical-to-basolateral direction. (A) Experimental setup. CRP was added to either the apical or basolateral chamber of Transwell with intact EC monolayers. CRP transcytosed to the opposite chambers were determined with ELISA. A-to-B, apical-to-basolateral; B-to-A, basolateral-to-apical. (B) Time and concentration-dependent transcytosis of CRP (n = 3). (C) Transcytosis of CRP across HAEC monolayers cultured on PET (BD Falcon, polyethylene terephthalate filters), PCF (Millipore, polycarbonates filters), and CM filters (Millipore, polytetrafluoroethylene filters) (n = 3). (D) Transcytosis of CRP across HUVEC, HCAEC, and HAEC monolayers (n = 3). (E) Effects of IFN-γ and hyperglycaemia on CRP transcytosis (n = 3). HAEC monolayers were cultured without (control) or with IFN-γ (103 U/mL, 12 h) or in the presence of high glucose (25 mM, 24 h). For clarity, the data points at different conditions are shifted along the Y-axis. (F) Lack of directionality of the transcytosis of Lucifer yellow, transported via paracellular route, across intact HACE monolayers (n = 3). Values are mean ± SEM. Figure 3 View largeDownload slide The bidirectional transcytosis of CRP prefers to apical-to-basolateral direction. (A) Experimental setup. CRP was added to either the apical or basolateral chamber of Transwell with intact EC monolayers. CRP transcytosed to the opposite chambers were determined with ELISA. A-to-B, apical-to-basolateral; B-to-A, basolateral-to-apical. (B) Time and concentration-dependent transcytosis of CRP (n = 3). (C) Transcytosis of CRP across HAEC monolayers cultured on PET (BD Falcon, polyethylene terephthalate filters), PCF (Millipore, polycarbonates filters), and CM filters (Millipore, polytetrafluoroethylene filters) (n = 3). (D) Transcytosis of CRP across HUVEC, HCAEC, and HAEC monolayers (n = 3). (E) Effects of IFN-γ and hyperglycaemia on CRP transcytosis (n = 3). HAEC monolayers were cultured without (control) or with IFN-γ (103 U/mL, 12 h) or in the presence of high glucose (25 mM, 24 h). For clarity, the data points at different conditions are shifted along the Y-axis. (F) Lack of directionality of the transcytosis of Lucifer yellow, transported via paracellular route, across intact HACE monolayers (n = 3). Values are mean ± SEM. 3.3 BM reverses the apical-to-basolateral preference of CRP transcytosis The in vitro observations would predict that circulating CRP can cross the endothelium and accumulate in tissues, as the result of more pronounced apical-to-basolateral transcytosis and abundance of CRP in the circulation. To test this prediction, we injected human CRP into mice and assessed CRP levels in the serum and aortic wall. Following a single intravenous injection, CRP was detectable in the aortic wall (Figure 4A), confirming CRP transcytosis in vivo. Unexpectedly, tissue CRP levels did not increase over time, instead they decreased in parallel with serum concentrations (Figure 4B). To address whether lack of detectable tissue accumulation of CRP was due to its short half-life (∼4 h) in the circulation of mice,18,19 we injected CRP every 4 h to maintain a constantly high circulating level (∼35 μg/mL) during the entire experiments. However, tissue accumulation of CRP remained undetectable (Figure 4A and B), suggesting that the apical-to-basolateral directional preference of CRP transcytosis observed in vitro is reversed in vivo. Figure 4 View largeDownload slide BM reverses the apical-to-basolateral preference of CRP transcytosis. (A and B) Mice received a single intravenous injection of human CRP (huCRP; 2.5 mg/kg) (n = 4) (left) or repeated injections every 4 h (n = 4) (right). The levels of CRP in the aortic tissue and serum were determined with immunoblotting (A) and ELISA (B), respectively. For immunoblotting, purified serum human CRP served as control (Ctrl). (C) A thick layer of Matrigel was coated onto Transwell filters before seeding HAECs to mimic BM present in the vessel wall. Experimental setup (left) and confirmation of formation of HAEC monolayers on Matrigel by confocal imaging (right). (D) CRP transportation across BM-coated Transwell filters in the absence of HAEC (n = 5). (E) Basolateral-to-apical directional preference of CRP transcytosis across HAEC monolayers on BM (n = 3). (F) Reversal of the directional preference depends on the thickness of the BM layer (n = 3–6). Preference index was calculated as (the amount of CRP transcytosed in the apical-to-basolateral direction)/(the amount of CRP transcytosed in the basolateral-to-apical direction). BM reversal of the directional preference of transcytosis of transferrin (Tf) (G) and low-density lipoproteins (LDL) across HAEC monolayers (H) (n = 3). Values are mean ± SEM. *P < 0.05 (Mann–Whitney’s U-test). Figure 4 View largeDownload slide BM reverses the apical-to-basolateral preference of CRP transcytosis. (A and B) Mice received a single intravenous injection of human CRP (huCRP; 2.5 mg/kg) (n = 4) (left) or repeated injections every 4 h (n = 4) (right). The levels of CRP in the aortic tissue and serum were determined with immunoblotting (A) and ELISA (B), respectively. For immunoblotting, purified serum human CRP served as control (Ctrl). (C) A thick layer of Matrigel was coated onto Transwell filters before seeding HAECs to mimic BM present in the vessel wall. Experimental setup (left) and confirmation of formation of HAEC monolayers on Matrigel by confocal imaging (right). (D) CRP transportation across BM-coated Transwell filters in the absence of HAEC (n = 5). (E) Basolateral-to-apical directional preference of CRP transcytosis across HAEC monolayers on BM (n = 3). (F) Reversal of the directional preference depends on the thickness of the BM layer (n = 3–6). Preference index was calculated as (the amount of CRP transcytosed in the apical-to-basolateral direction)/(the amount of CRP transcytosed in the basolateral-to-apical direction). BM reversal of the directional preference of transcytosis of transferrin (Tf) (G) and low-density lipoproteins (LDL) across HAEC monolayers (H) (n = 3). Values are mean ± SEM. *P < 0.05 (Mann–Whitney’s U-test). In blood vessels, endothelial cells are attached to a layer of fibrous extracellular matrix, termed BM.20 To mimic the physiological situation, we seeded HAECs onto Transwell filters coated with a thick layer of Matrigel (Figure 4C). CRP diffusion through Matrigel-coated filters did not show any directional preference (Figure 4D). As anticipated, HAECs formed a confluent monolayer on Matrigel. Surprisingly, the directional preference of CRP transcytosis was reversed to the basolateral-to-apical direction under this experimental condition (Figure 4E). It is unlikely that such reversal was due to effects of BM constituents on HAECs since a thin layer of collagen was also applied on top of Matrigel similar to that done in experiments described above. The extent of reversal appeared to depend on the thickness of the Matrigel layer (Figure 4F). Thus, increasing the thickness resulted in a gradual shift of CRP transcytosis from an apical-to-basolateral dominance to a basolateral-to-apical dominance. Importantly, we detected a similar reversal in directional preference for transcytosis of transferrin (Figure 4G) and LDL (Figure 4H) with the presence of Matrigel layer. These findings would indicate that BM regulates the directionality of protein transcytosis through a general mechanism, possibly related to certain physical aspect of BM. 3.4 BM sieving enforces immediate retrograde transcytosis A fundamental feature of BM is the dense, mesh-like 3D organization,20 inherently resembling a sieve at nanoscale that hinders the movement of large molecules. We thus hypothesized that following transcytosis from the apical compartment, the diffusion of CRP away from the basolateral EC surface would be slowed by BM sieving, thereby leading to a high local concentration that, in turn, would promote retrograde transportation. We found that Matrigel coated on filters exhibited a mesh-like organization (Figure 5A) and markedly retarded the diffusion of CRP (Figure 5B). Indeed, the rate of CRP exchange across BM layer was reduced by about three orders of magnitude (Figure 5B). Next, we visualized the diffusion pattern of CRP in BM following apical-to-basolateral transcytosis across HAEC monolayers (Figure 5C) and noted two features of the concentration gradient of transcytosed CRP: a sharp diffusion front manifesting the retarded diffusion in BM and a shallow valley just beneath the basolateral surface of HAEC, where the peak of CRP concentration was expected. We detected similar patterns with transferrin (Figure 5D). Figure 5 View largeDownload slide BM sieving enforces retrograde transportation to counteract apical-to-basolateral transcytosis. (A) Mesh-like organization of Matrigel layer coated on Transwell filters visualized with scanning electron microscopy (n = 5). (B) Reduced CRP diffusion in apical-to-basolateral (A to B) and basolateral-to-apical (B to A) directions across Transwell filters coated with Matrigel at varying thickness (n = 5). (C) CRP diffusion in BM following apical-to-basolateral transcytosis across HAEC monolayers. FITC-labeled CRP (50 μg/mL) was added to the apical chamber of Transwell for 30 min. Representative 3D reconstruction (upper panel) and the intensity profiles along the Z-axis (lower panel) are shown. The shallow valley beneath HAEC monolayers suggests a pronounced retrograde transportation that disperses the accumulation of CRP transcytosed from the apical side. This valley was not observed in BM without HAEC monolayer. Representative images are shown (n = 5). (D) Transferrin diffusion in BM following apical-to-basolateral transcytosis across HAEC monolayers (n = 3). (E) Endocytosis of CRP at the basolateral surface of HAEC monolayer following apical-to-basolateral transcytosis. Alexa-568-labeled CRP (50 μg/mL) was allowed to bind the apical surfaces of HAEC monolayers at 4°C for 25 min, followed by washing and transcytosis at 37°C for 5 min (left). TIRF imaging identified events of CRP endocytosis at the basolateral surfaces (right) (n = 5). (F) Decreased CRP diffusion across Transwell filters coated with agarose at varying thickness (n = 5). (G) Reversal of the directional preference of CRP transcytosis across agarose-coated Transwell filters (n = 3). Values are mean ± SEM. Figure 5 View largeDownload slide BM sieving enforces retrograde transportation to counteract apical-to-basolateral transcytosis. (A) Mesh-like organization of Matrigel layer coated on Transwell filters visualized with scanning electron microscopy (n = 5). (B) Reduced CRP diffusion in apical-to-basolateral (A to B) and basolateral-to-apical (B to A) directions across Transwell filters coated with Matrigel at varying thickness (n = 5). (C) CRP diffusion in BM following apical-to-basolateral transcytosis across HAEC monolayers. FITC-labeled CRP (50 μg/mL) was added to the apical chamber of Transwell for 30 min. Representative 3D reconstruction (upper panel) and the intensity profiles along the Z-axis (lower panel) are shown. The shallow valley beneath HAEC monolayers suggests a pronounced retrograde transportation that disperses the accumulation of CRP transcytosed from the apical side. This valley was not observed in BM without HAEC monolayer. Representative images are shown (n = 5). (D) Transferrin diffusion in BM following apical-to-basolateral transcytosis across HAEC monolayers (n = 3). (E) Endocytosis of CRP at the basolateral surface of HAEC monolayer following apical-to-basolateral transcytosis. Alexa-568-labeled CRP (50 μg/mL) was allowed to bind the apical surfaces of HAEC monolayers at 4°C for 25 min, followed by washing and transcytosis at 37°C for 5 min (left). TIRF imaging identified events of CRP endocytosis at the basolateral surfaces (right) (n = 5). (F) Decreased CRP diffusion across Transwell filters coated with agarose at varying thickness (n = 5). (G) Reversal of the directional preference of CRP transcytosis across agarose-coated Transwell filters (n = 3). Values are mean ± SEM. The observed valley could be explained by a pronounced retrograde transcytosis that outcompeted the apical-to-basolateral transcytosis. Consistently, TIRF imaging confirmed CRP endocytosis at the basolateral surface of HAECs (Figure 5E). Since the apical-to-basolateral transcytosis was the only source of CRP beneath HAECs in our experimental setting, the endocytosed CRP should be the result of immediate retrograde transportation of exocytosed CRP. Of note, the events of exocytosis and endocytosis appeared to be spatially and temporally coupled: the latter usually happened shortly after and in proximity to the former. These results would suggest that BM regulates transcytosis by physical modulation of ligand diffusion rather than by biological modulation of cellular responses. Coating filters with agarose gel, another sieve-like matrix, also markedly retarded the diffusion of CRP (Figure 5F) and diminished the apical-to-basolateral preference of CRP transcytosis (Figure 5G). Therefore, BM sieving-enforced retrograde transcytosis in situ may be a key determinant that restricts tissue accumulation of circulating CRP. While BM is an integral part of vasculature, it is incomplete/fenestrated in certain types of small vessels, such as liver sinusoidal capillaries.1,21 If BM sieving is critical in determining tissue accumulation of circulating proteins, CRP accumulation in organs would differ as per the completeness of BM in their vessels. Injection of Alexa-568-labeled CRP into mice resulted in the presence of CRP in the aorta where it primarily colocalized with laminin-marked BM beneath the EC layer (Figure 6A). In contrast, strong diffuse CRP signals were detected in the liver, showing no enrichment at the BM (Figure 6B). In the lung, which has abundant microcirculation and complete BM, however, only weak signals of CRP could be detected (Figure 6C). Consistently, the Pearson’s correlation coefficients between CRP and laminin were higher in the aorta and lung but was close to zero in the liver (Figure 6E). However, much higher quantities of CRP were accumulated in the liver than in other organs (Figure 6F). Collectively, these findings corroborate the role of BM sieving in the regulation of tissue accumulation of circulating proteins. Though fenestrae (pores) on liver sinusoidal endothelia might confound the interpretation, it should be noted that a large portion of liver sinusoidal endothelia is not fenestrated. Moreover, glomerular capillaries have fenestrae,1,21,22 but possess complete BM. Importantly, we detected increased colocalization of CRP with BM, but reduced accumulation in glomeruli (Figure 6D–F). Figure 6 View largeDownload slide Tissue accumulation of circulating CRP correlates to BM completeness. Mice were injected with Alexa-568-labeled CRP (2.5 mg/kg, intravenous) and 40 min later selected tissues were prepared for assessment of CRP accumulation. Representative images of the aorta (A), liver (B), lung (C), and glomerulus (D) (n = 6–13). Laminin was used to mark BM. Liver sinusoid are indicated with asterisk. Quantification of CRP colocalization with laminin (E) and CRP tissue accumulation (F) (n = 6–13). The boxes indicate the interquartile range between the 25th and 75th percentiles. *P < 0.05, ***P < 0.001 (Kolmogorov–Smirnov test). Figure 6 View largeDownload slide Tissue accumulation of circulating CRP correlates to BM completeness. Mice were injected with Alexa-568-labeled CRP (2.5 mg/kg, intravenous) and 40 min later selected tissues were prepared for assessment of CRP accumulation. Representative images of the aorta (A), liver (B), lung (C), and glomerulus (D) (n = 6–13). Laminin was used to mark BM. Liver sinusoid are indicated with asterisk. Quantification of CRP colocalization with laminin (E) and CRP tissue accumulation (F) (n = 6–13). The boxes indicate the interquartile range between the 25th and 75th percentiles. *P < 0.05, ***P < 0.001 (Kolmogorov–Smirnov test). 3.5 Tissue contribution to circulating CRP Given the limited accumulation of circulating CRP into most tissues, it appears plausible that in situ generation may be the major source for local CRP. Next, we studied whether locally produced CRP could conversely be transported into the circulation by examining the redistribution of tissue-accumulated, Alexa-568-labeled CRP following in vivo perfusion with PBS (Figure 7A–C). Perfusion markedly reduced CRP signals in the aorta and lung in parallel with reduced colocalization of CRP with BM (Figure 7D and E). These would suggest that CRP accumulated at the BM was ready for leaving the aorta via retrograde transcytosis, rather than diffusing deep into the tissues. In contrast, CRP signals were evenly reduced across the entire liver section, indicating a degradation-dominated reduction. To corroborate the contribution of locally produced CRP to circulating CRP level, we inoculated mice with CT26 colon carcinoma cells expressing a human CRP vector. Human CRP was soon detectable in the serum, and its concentration gradually increased over time (Figure 7F). Figure 7 View largeDownload slide Local production of CRP may contribute to serum CRP levels. Mice were injected with Alexa-568-labeled human CRP (2.5 mg/kg, intravenous). Forty minutes later, the animals were euthanized, perfused with PBS for 1.5 h and then selected tissues were prepared for assessment of CRP accumulation. Representative images of the aorta (A), liver (B), and lung (C) (n = 4–5). Liver sinusoids are indicated with an asterisk. Quantification of CRP colocalization with laminin (D) and CRP tissue accumulation (E). The boxes indicate the interquartile range between the 25th and 75th percentiles. (F) Tumour formation (left) and serum levels of human CRP (right) in mice inoculated with CT26 colon carcinoma cells expressing a huCRP vector or an empty vector (n = 3–6). Values are mean ± SEM, *P < 0.05, **P < 0.01 (Kolmogorov–Smirnov test). Figure 7 View largeDownload slide Local production of CRP may contribute to serum CRP levels. Mice were injected with Alexa-568-labeled human CRP (2.5 mg/kg, intravenous). Forty minutes later, the animals were euthanized, perfused with PBS for 1.5 h and then selected tissues were prepared for assessment of CRP accumulation. Representative images of the aorta (A), liver (B), and lung (C) (n = 4–5). Liver sinusoids are indicated with an asterisk. Quantification of CRP colocalization with laminin (D) and CRP tissue accumulation (E). The boxes indicate the interquartile range between the 25th and 75th percentiles. (F) Tumour formation (left) and serum levels of human CRP (right) in mice inoculated with CT26 colon carcinoma cells expressing a huCRP vector or an empty vector (n = 3–6). Values are mean ± SEM, *P < 0.05, **P < 0.01 (Kolmogorov–Smirnov test). 4. Discussion Our results identify a novel physical mechanism by which the BM modulates the directionality of transcytosis and consequently contributes to regulating tissue accumulation of circulating proteins. While serum proteins, such as CRP, transferrin, and LDL, can undergo transcytosis from the circulation into the blood vessel wall, their tissue accumulation is limited by BM sieving-enforced retrograde transportation. This mechanism is supported by several lines of evidence. Firstly, BM exhibits mesh-like organization resembling a sieve at nanoscale (Figure 5A) that markedly retards the diffusion of CRP (Figure 5B). Secondly, the retarded diffusion accumulates CRP in BM beneath endothelial cells following transcytosed from the apical compartment (Figure 5C). Thirdly, the accumulated CRP undergoes immediate endocytosis at the basolateral surfaces of endothelial cells (Figure 5C, E), thereby counteracting the apical-to-basolateral transcytosis. These findings could explain BM reversal of the directionality of CRP transcytosis, and would predict that this action largely depends on the physical properties of BM. In line with this prediction, we demonstrate that BM reversal of the directionality of CRP transcytosis depends on matrix thickness and porosity (Figures 4F and 5G), but not on matrix composition (Figures 4C, E and 5G). Moreover, BM also reverses the directionality of transferrin and LDL, which possess biological properties different from that of CRP. Besides being a nonspecific marker of inflammation,4,5 over 50 prospective clinical studies have reported that minor elevations in the baseline serum CRP level (1∼3 μg/mL) are independently associated with increased risk of cardiovascular disease in apparently healthy subjects.23 The predictive power of CRP is comparable to or even superior than that of total cholesterol or elevated blood pressure.23,24 CRP has also been detected in initial atherosclerotic lesions and advanced plaques6–8 where it may contribute to disease progression.4,5 An important yet not fully addressed issue is the origin of plaque-localized CRP. Our data demonstrate opposing forces of CRP transcytosis and BM sieving-enforced retrograde transportation in regulating its tissue accumulation. CRP transcytosis is mediated partly through FcγR1A and involves clathrin-dependent endocytosis and aligning CRP-containing vesicles with microtubules, the major tracks for long-range cellular transportation.3 The BM sieving-enforced retrograde transportation would easily explain the absence of CRP in normal blood vessels6,7 and the lack of infiltration of 125I-labelled, circulating CRP into inflamed tissues.9 In contrast, lack of tissue CRP accumulation cannot be attributed to efficient local degradation of CRP, for it is catabolized by hepatocytes in vivo,25 or to its rapid clearance from the vessel wall, for the cycling rate of interstitial fluids is several orders slower than that of blood. A critical implication of BM sieving-enforced retrograde transportation is that CRP present in tissues is predominantly produced in situ, rather than originating from the circulation via transcytosis or attached to transmigrating leucocytes.26 Enhanced CRP expression has been reproducibly demonstrated in a variety of lesions, including atherosclerotic plaques27–30 and tumours.31 CRP formation has also been detected in cell types other than hepatocytes,32–44 including cells of the vascular wall.32–37 Here, we further show that tissue-generated CRP can be transported into the blood via retrograde transcytosis. This would argue that CRP produced locally in plaques could cross the intact endothelial barrier and be released into the circulation, resulting in higher circulating levels downstream to lesions as observed in many previous studies.29,30,45,46 At the sites of damaged endothelial barrier due to plaque rupture or erosion, excessive accumulation of circulating CRP in lesions might, however, occur via direct deposition, resulting in lower serum CRP at these sites.47 Hence, it appears plausible that CRP produced by and released from atherosclerotic plaques might contribute to elevated serum CRP levels, and therefore to cardiovascular risk prediction. These findings also highlight the potential pathological importance of locally generated CRP in addressing the role of CRP in inflammation4 as well as the long-standing controversy of whether CRP is causally involved in atherogenesis.23 Our results with pharmacological blockade of FcγR1A imply a role for this receptor in mediating CRP transcytosis. Lack of statistically significant increases in CRP transcytosis following FcγR1A overexpression may reflect pathway saturation, functional redundancy among FcγR or receptor-dependent or -independent internalization pathways (e.g. macropinocytosis). FcγR1A and 2B have been identified as functional receptors for native CRP on several cell types, including endothelial cells,15 whereas monomeric CRP predominantly binds to FcγR3.48,49 Native CRP is the major conformation secreted by most cell types, though direct release of mCRP has also been reported in some cell types.50 We have previously shown that mCRP injected into the circulation rapidly redistributes into tissues12,19 likely via a lipid raft-mediated mechanism.12,51 Trafficking of mCRP produced locally within the inflammatory microenvironment into the circulation remains, however, to be investigated. Although it has been well recognized that transcytosis across endothelial barrier is bidirectional, the net transportation of circulating proteins is believed to be in apical-to-basolateral direction due to the intuitive premise of plasma protein concentration gradients from the blood to the interstitium.2 This gradient coupled with the slow cycling rate of interstitial fluids would imply that strong tissue accumulation of circulating proteins is inevitable, however, this assumption appears to be incorrect under both physiological and most pathological conditions. Here, we propose that BM beneath the basolateral surface of endothelial barrier could flatten the protein gradient. Indeed, the sieve-like structure of BM would hinder further diffusion of circulating proteins that were transcytosed from the blood, thereby enforcing high local levels at a confined space to promote retrograde transportation. This would lead to a dynamic equilibrium between protein influx due to apical-to-basolateral transcytosis and efflux by retrograde transcytosis and limited diffusion. Consequently, excessive tissue accumulation of circulating proteins including CRP, transferrin, and LDL, is limited. This mechanism depends solely on the physical features of the BM and protein substrates (e.g. size), and therefore can, in principle, be applied universally to regulation of blood-tissue exchange of blood-borne proteins across the intact endothelial barrier. In summary, our study identifies a novel physical mechanism by which the BM modulates the directionality of protein transcytosis and consequently contributes to maintaining tissue homeostasis. Conflict of interest: none declared. Funding This work was supported by the National Natural Science Foundation of China [31770819, 31671339, 31570749, 31470718]; the Fundamental Research Funds for the Central Universities [lzujbky-2016-k11]; the China Postdoctoral Science Foundation [2018T111039, 2016M602799]; and the Natural Science Foundation of Shaanxi Province [2017JQ3026]. Footnotes Time for primary review: 18 days References 1 Augustin HG , Koh GY. Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology . Science 2017 ; 357 : eaal2379. Google Scholar Crossref Search ADS PubMed 2 Tuma P , Hubbard AL. Transcytosis: crossing cellular barriers . Physiol Rev 2003 ; 83 : 871 – 932 . Google Scholar Crossref Search ADS PubMed 3 Mehta D , Malik AB. Signaling mechanisms regulating endothelial permeability . Physiol Rev 2006 ; 86 : 279 – 367 . Google Scholar Crossref Search ADS PubMed 4 Wu Y , Potempa LA , El Kebir D , Filep JG. 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TI - Matrix sieving-enforced retrograde transcytosis regulates tissue accumulation of C-reactive protein
JF - Cardiovascular Research
DO - 10.1093/cvr/cvy181
DA - 2019-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/matrix-sieving-enforced-retrograde-transcytosis-regulates-tissue-r0o2s0XNqc
SP - 440
VL - 115
IS - 2
DP - DeepDyve
ER -