Chemokines in atherosclerotic lesion development and stability : from mice to man Jager, S.C.A. de

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(1)Chemokines in atherosclerotic lesion development and stability : from mice to man Jager, S.C.A. de. Citation Jager, S. C. A. de. (2008, October 23). Chemokines in atherosclerotic lesion development and stability : from mice to man. Faculty of Science, Leiden University|Department of Biopharmaceutics, Leiden Amsterdam Center for Drug Research. Retrieved from https://hdl.handle.net/1887/13158 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/13158. Note: To cite this publication please use the final published version (if applicable)..

(2) 9. Perivascular Mast Cells Promote Atherogenesis and Induce Plaque Destabilization in ApoE Deicient Mice. Saskia C.A. de Jager1*, Ilze Bot1*, Alma Zernecke2, Ken A. Lindstedt3, Theo J.C. van Berkel1, Christian Weber2 and Erik A.L. Biessen1. Circulation. 2007;115(19):2516-25. Abstract Background: Mast cells are major effector cells in allergy and host defence responses. Their increased number and state of activation in perivascular tissue during atherosclerosis may point to a role in cardiovascular disorders. In this study, we investigated the contribution of perivascular mast cells to atherogenesis and plaque stability in ApoEdeicient mice. Methods and Results: We show here that episodes of systemic mast cell activation during plaque progression in mice leads to robust plaque expansion. Targeted activation of perivascular mast cells in advanced plaques sharply increases the incidence of intraplaque hemorrhage, macrophage apoptosis, vascular leakage and CXCR2/VLA-4mediated recruitment of leukocytes to the plaque. Importantly, treatment with the mast cell stabilizer cromolyn does prevent all of the adverse phenomena elicited by mast cell activation. Conclusions: This is the irst study to demonstrate that mast cells play a crucial role in plaque progression and destabilization in vivo. We propose that mast cell stabilization could be a new therapeutic approach to the prevention of acute coronary syndromes. 139.

(3) Chapter 9. Introduction Acute coronary syndromes including unstable angina and myocardial infarction are commonly caused by erosion or rupture of vulnerable atherosclerotic plaques13 . Inlammatory cells are considered to play a key role in the pathogenesis of plaque rupture4-6. Indeed, one of the inlammatory cell types, the mast cell7, 8, has been shown to accumulate in the rupture-prone shoulder region of human atheromas9. Activated mast cells containing proteases such as tryptase and chymase have been identiied at the site of rupture in specimens of human coronary arteries10-13. Human coronary artery specimens contain TNFα-rich activated mast cells14, 15, which potentially aggravate the ongoing inlammatory response and may ultimately lead to plaque destabilization16. Not only intimal inlammation but also inlammation of the arterial adventitia has been shown to inluence the plaque vulnerability17. Activated mast cells have been identiied in the adventitia of vulnerable and ruptured lesions in patients with myocardial infarction18-20 and more importantly, their number was found to correlate with the incidence of plaque rupture and erosion19. However, it remains to be clariied whether adventitial mast cells are actively modulating lesion composition and are instrumental in causing plaque rupture or rather part of the inlammatory cell recruitment secondary to plaque rupture. In this study, we demonstrate that systemic mast cell activation during atherogenesis leads to increased plaque progression. Moreover, we show that local activation of mast cells in the adventitia of advanced carotid artery plaques promotes macrophage apoptosis, microvascular leakage and de novo leukocyte inlux. Importantly, this culminates in a greatly enhanced incidence of intraplaque hemorrhage. Finally, mast cell stabilization by cromolyn was seen to prevent these pathophysiological events by inhibition of mast cell degranulation. Methods Experimental Design All animal work was performed in compliance with the Dutch government guidelines. Male ApoE-/- mice (26 weeks old), obtained from the local animal breeding facility, were fed a Western type diet, containing 0.25% cholesterol and 15% cacaobutter (SDS, Sussex, UK). After 4 weeks of Western-type feeding, all isolurane anaesthetized animals were skin-sensitized on day 1 and 2 with dinitroluorobenzene (DNFB, 0.5% v/v, Janssen Chimica, Beerse, Belgium) or vehicle control solution (acetone:olive oil 4:1, n=7 per group) as described by Kraneveld et al.21. On day 5, the mice were challenged intravenously by injection of dinitrophenyl-albumin (DNP, 1 mg/animal), which was repeated once weekly for another two weeks to induce systemic adventitial mast cell activation. A separate group of mice received an intraperitoneal injection of the mast cell stabilizer cromolyn (50 mg/kg, Sigma, Zwijndrecht, The Netherlands)22, 23 30 minutes before and after DNP challenge. After 8 weeks of diet feeding, the mice were anaesthetized. In situ ixation through the left cardiac was performed24 and brachiocephalic artery lesions were analyzed. To determine the effect of local adventitial mast cell activation on advanced atherosclerotic lesions, carotid artery plaque formation was induced by perivascular collar placement in male apoE-/- mice as described previously24. Mice were anaesthetized by subcutaneous injection of ketamine (60 mg/kg, Eurovet Animal Health, Bladel, The Netherlands), fentanyl citrate and luanisone (1.26 mg/kg and 2 mg/kg respectively, Janssen Animal Health, Sauderton, UK). Five weeks after collar placement all animals were skin-sensitized as described above (control: n=14, DNFB: n=13). On day 5, the mice were challenged perivascularly by applying pluronic F-127 gel (25% w/v) or pluronic F127 gel containing DNP (50 μg/animal) at the lesion site. To measure de novo iniltration of circulating leukocytes into the lesions, some of the mice were injected intravenously 140.

(4) Adventitial Mast Cells Induce Plaque Destabilization 25. with Rhodamine-6G (0.67 mg/kg) to label circulating leukocytes. In a separate experimental set-up, two groups of mice (control: n=11 and DNFB: n=10) received an intravenous injection containing 25 mg/kg of cromolyn thirty minutes before local DNP or control challenge and twice daily during challenge by intraperitoneal injections with 50 mg/kg of cromolyn. Three days after challenge the animals were anaesthetized and in situ perfusion-ixation was performed, after which the carotid artery lesions were analyzed. Histology Mast cells were visualized by staining of 5 μm cryosections with aqueous toluidin blue (Sigma), while mast cell phenotype was established using an Alcian Blue/Safranin O staining (Sigma). Neutrophils were stained with naphthol AS-D chloroactetae esterase (Sigma). Iron staining was performed according to Perl’s method. Macrophage content of the lesions was assessed using a rat monoclonal MoMa-2 antibody (Serotec, Kidlington, Oxford, UK). Endothelium was stained by use a CD31 monoclonal antibody (BD BioSciences), while apoptosis was visualized using a terminal deoxytransferase dUTP nick-end labeling (TUNEL) kit (Roche Diagnostics). Morphometry Morphometric analysis (Leica Qwin image analysis software) was performed on hematoxylin-eosin stained sections of brachiocephalic artery lesions at 150 μm from the bifurcation and of the carotid arteries at the site of maximal stenosis24. Toluidin blue stained sections were used for histological examination for the presence of adventitial mast cells. Mast cells numbers, the extent of mast cell degranulation and presence of iron were assessed manually. MoMa-2 and TUNEL positive areas were quantiied by Leica Qwin image analysis software and in addition, TUNEL positive nuclei were counted manually. All morphometric analyses were performed by blinded independent operators (S.C.A.d.J./I.B.). Cell culture MC/9 cells26, kindly provided by Dr. Renauld from the Ludwig Institute for Cancer Research in Belgium, were cultured as described previously27. MC/9 cells (2.5*105 cells/ mL) were degranulated by incubation with 0.5 μg/mL of compound 48/80 (Sigma) for 15 minutes at 37 °C. Cells were centrifuged (1500 rpm, 5 minutes) and the supernatant was used for further experiments. Bone marrow derived mast cells (BMMCs) from C57Bl/6 mice were cultured as previously described28 and degranulated with compound 48/80 as described above. Peritoneal mast cells (PMCs) from C57Bl/6 mice were isolated by lavage of the peritoneal cavity with 10 mL of ice-cold PBS. Cells were seeded as 2*106 cells/mL in RPMI containing 10% FBS 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and mIL3 and allowed to attach for 1 hour. Non-adhered cells were seeded at 2.5*105 cells/mL and used for degranulation experiments. To determine β-hexosaminidase activity in the releasate, MC/9 cells or PMCs were degranulated as described above in HEPES-tyrode supplemented with 0.1 % fatty acid free BSA (Sigma). 50 μL of supernatant was added to 50 μL 2 mM 4-nitrophenyl Nacetyl-b-D-glucosaminide (Sigma) in 0.2 M citrate (pH 4.5) and incubated at 37 °C for 2 hours. After addition of 150 μL 1 M Tris (pH 9.0), absorbance was measured at 405 nm. To measure chymase and tryptase release after degranulation, 50 μL supernatant was added to 2 mM S-2288 (tryptase substrate, Chromogenix, Lexington, USA) or S-2586 (chymase substrate, Chromogenix) in PBS supplemented with 100 U/mL heparin. After 2 hours (tryptase) or 48 hours (chymase) at 37 °C, OD405 was measured. Histamine and VEGF levels were measured with a Histamine ELISA (Neogen, Lansing, MI) and a VEGF ELISA (Biosource, Etten-Leur, The Netherlands) according to manufacturers instructions. 2.5*105 MC/9 cells or PMCs were lysed in 1 mL 2% Triton in HEPES-tyrode 141.

(5) Chapter 9. and used for total release in each assay and supernatant of non-degranulated mast cells was used as control. Apoptosis assay VSMCs were obtained from thoracic aortas from male C57Bl/6 mice by collagenase digestion and cultured as previously described29. The murine macrophage cell line RAW 264.7 was cultured in DMEM containing 10% FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Cambrex, Verviers, Belgium). VSMCs and RAW 264.7 were seeded at a density of 105 cells/cm2 and exposed to supernatant from degranulated and undegranulated MC/9 cells for 24 hours (RAWs) or for 48 and 72 hours (vSMCs). After incubation, cells were stained with propidium iodide (Sigma) and RNase H (Boehringer Mannheim, Mannheim, Germany) was added to avoid RNA contamination in the measurements. DNA fragmentation was measured by FACS analysis (FACScalibur, BD Biosciences). The effect of mast cell chymase and tryptase inhibition on apoptosis was determined using the soybean trypsin inhibitor and leupeptin (both 100 mg/L, Sigma)30, while the involvement of histamine receptors in apoptosis was addressed by measuring apoptosis in the presence of the H1-, H2- and H3-receptor antagonists triprolide (1 μM), cimetidine (100 μM) and thioperamide (1 μM, all from Sigma)31 respectively. To determine the effect or primary mast cells on macrophage apoptosis, peritoneal macrophages were exposed to supernatant of BMMCs and PMCs, degranulated with compound 48/80 similarly as described for RAW 264.7 cells. The effect of single mast cell constituents on macrophage apoptosis was addressed by pre-incubating RAW 264.7 cells with either histamine (100 μM) or tryptase (500 U/L, both from Sigma) for 16 hours and after removal of this medium, incubating the macrophages with either tryptase (500 U/L) or histamine (100 μM) for an additional 6 hours, after which apoptosis was measured. Proliferation assay The murine endothelial cell line H5V was cultured in DMEM containing 10% FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Cambrex) and were seeded at a density of 1*104 cells per well. The cells were allowed to attach and serum starved in medium containing 1% FBS to synchronize cell cycle. The next day, the H5V cells were exposed to mast cell supernatants and incubated overnight with 0.5 μCi [3H]thymidine (Amersham, Uppsala, Sweden) per well. [3H]Thymidine incorporation was quantiied in a liquid scintillation counter (Packard, USA). C57Bl/6 and Kit(W-sh/W-sh) mice To determine whether cromolyn had any side-effects on other cell types besides mast cells, we skin sensitized C57Bl6 mice (Charles River Laboratories, Maastricht, The Netherlands) with either DNFB or vehicle control solution. The mice were systemically challenged with PBS or DNP and treated with cromolyn as described above (n=5 per group). At 4 weeks after sensitization, the mice were sacriiced and sensitized skin, peritoneal cells and serum was analyzed. Similarly, C57Bl6 mice or mast cell deicient Kit(W-sh/W-sh) mice32 were skin sensitized, locally challenged at the common carotid artery and a subset of mice was treated with cromolyn as described previously (n=5 per group). At three days after challenge, the mice were sacriiced and sensitized skin, peritoneal cells and serum were analyzed. Skin cryosections of the mice (5 μm) were stained with Wright’s Giemsa (Sigma) and cell differentiation at 1000x magniication was performed by a blinded independent operator (I.B.). Peritoneal cells were stained with α-CD117 (mast cells), α-GR1 (neutrophils), α-CD11b (macrophages) and α-CD3 (T-cells, all from Immunosource, Halle-Zoersel, Belgium) and cell differentiation was determined using FACS analysis (FACScalibur). Myeloperoxidase (MPO) activity was measured to determine neutrophil activity. In short, 5*105 peritoneal cells were lysed in 150 μL 50 mM Tris/0.05% Triton and 50 μL was added to 50 μL of substrate (0.1 mg/ mL TMB, 0.003% H2O2 and 13 μg/mL resorcinol in 10 mM citrate, pH 5) and incubated 142.

(6) Adventitial Mast Cells Induce Plaque Destabilization. at room temperature for 15 minutes. After addition of 100 μL 2 M H2SO4, absorbance was measured at 450 nm. 50 mM Tris/0.05% Triton was used as negative control. Serum IL-2, IL-4 and IFNγ levels were measured using a mouse Th1/Th2 ELISA kit (eBioscience, San Diego, USA) and TNFα serum levels were analyze with a mouse TNFα ELISA (BD, Breda, The Netherlands). IgE levels were measured after systemic challenge using an IgE ELISA (Bethyl Laboratories, Montgomery, USA). Microvascular leakage Increased microvascular permeability was assessed essentially as described by Sirois et al.33 and Walls et al.34 with minor modiications. In short, male C57Bl/6 mice were injected intradermally at randomized sites with either 5*105 MC/9 mast cells suspended in PBS containing 50 μg/mL compound 48/80 in the absence of presence of histamine receptor antagonists (0.1 mM triprolidine, 10 mM cimetidine or 0.1 mM thioperamide) or chymase and tryptase inhibitors (10 g/L SBTI or leupeptin, respectively). Immediately after intradermal injection of the cell suspensions, 100 μL 1.25% Evans Blue was injected intravenously and after 30 minutes, the surface area of Evans Blue stained skin was measured. To measure de novo iniltration of circulating leukocytes into the skin, mice were injected intravenously with Rhodamine-6G as described above. After 30 minutes, the skin was ixed and cellular iniltrates were scored manually. Intravital Microscopy ApoE-/- mice were sensitized and locally challenged at ive weeks after collar placement as described above and at three days after challenge, circulating leukocytes were stained in vivo by intravenous injection of 100 μL 0.02% Rhodamine-6G. After 30 minutes, video microscopy of the common carotid artery proximal to the collar and towards the shoulder of the plaque was performed using a Zeiss Axiotech microscope (water immersion objectives: 20x /0.5 W, 10x /0.3 W; Zeiss, Munich, Germany) with a HBO 100 mercury lamp for epi-illumination (Leistungselektronik Jena GmbH, Jena, Germany). The number of cells adherent to the vessel wall was quantiied in at least 3 high power ields (x20 objective) per artery and representative images were recorded using overview ields (x10 objective). Ex vivo perfusion of carotid arteries Three days after the local DNP challenge as previously described, the carotid arteries from apoE-/- mice were isolated, transferred onto a microscope stage (saline immersion objective) and perfused with Mono Mac6 cells (106/mL) labeled with Calcein-AM (Invitrogen, Breda, The Netherlands) in MOPS-buffered physiological salt solution at 4 μL/min35. Cells were left untreated or pretreated with isotype control, mAb against CXCR2 (10 μg/mL, MAB311, R&D Systems), VLA-4 (10μg/mL, HP2/1, Immunotech) or Met-RANTES (1 μg/mL, kindly provided by Peter Nelson, University of Munich). Adhesive interactions with the vessel wall were recorded using stroboscopic epi-luorescence illumination (Drelloscop 250, Drello) and irmly adherent cells were counted after 10 min. Statistical analysis Data are expressed as mean ± SEM. A 2-tailed Student’s t-test was used to compare individual groups, while multiple groups were compared with a one-way ANOVA and a subsequent Student-Newman-Keuls multiple comparisons test. Non-parametric data were analyzed using a Mann-Whitney U test. Frequency data analysis was performed by means of the Fisher’s exact test. Matched non-parametric data were analyzed with the Friedman test. A level of p<0.05 was considered signiicant.. 143.

(7) Chapter 9. Results Systemic mast cell activation and plaque morphometry First, we delineated the effect of systemic mast cell activation during atherosclerotic lesion progression in apoE-/- mice. In the adventitia of the brachiocephalic artery (BCA) lesions, the percentage of activated mast cells at 7 days after mast cell activation was 62 ± 9% in controls, 75 ± 7% in DNP challenged mice and 54 ± 10% in DNP challenged mice that were treated with the mast cell stabilizer cromolyn (p=Not Signiicant (NS)). Despite the fact that mast cells had only been activated for three times during eight weeks of lesion development, the BCA lesion size of DNP challenged mice was signiicantly increased by >2-fold compared to controls (73 ± 11*103 μm2 versus 35 ± 11*103 μm2, p<0.05, Figure 1A and B). Interestingly, treatment of DNP-challenged animals with the mast cell stabilizer cromolyn prevented the DNP-induced plaque expansion (42 ± 8*103 μm2, P<0.05 compared to DNP-challenged mice). Relative macrophage content (MoMa2 staining) did not differ between the groups (data not shown), while also the amount of adventitial neutrophils was unchanged (20 ± 1 in control mice, 19 ± 5 in DNP challenged mice and 16 ± 1 neutrophils/section in cromolyn treated DNP challenged mice, p=NS). A. 100000. P. P. 2. Plaque Size (Mm ). 80000. 60000. 40000. 20000. 0. Control. DNP. DNP + cromolyn. B. #ONTROL. $.0. $.0 CROMOLYN. Figure 1: Effect of systemic mast cell activation on brachiocephalic artery lesions. (A) Lesion analysis of brachiocephalic artery lesions of systemically challenged apoE-/- mice, which shows that DNP challenge with concomitant mast cell activation aggravated atherosclerotic lesion progression compared to control challenged animals. Cromolyn treatment during systemic DNP challenge normalized the DNP induced lesion progression. (B) Representative pictures of a control brachiocephalic artery lesion of an apoE-/- mouse (left panel), a DNPchallenged animal (middle picture) and that of a DNP-challenged animal that had received cromolyn during challenge (right panel).. Local adventitial mast cell activation and plaque morphology Since mast cell density and activation is particularly high in the adventitia of human type V/VI atherosclerotic lesions and as we cannot exclude a systemic inlammatory response when mast cells are activated as described above, we assessed the inluence of focal and acute adventitial mast cell activation on advanced atherosclerotic plaques in ApoE/mice. The hapten challenge was therefore applied perivascularly by administering a DNP loaded pluronic F-127 gel at the collar-induced carotid artery lesion24. Morphometric analysis of the lesions did not reveal any differences in plaque size between control and DNP-challenged animals at three days after challenge (54 ± 8*103 μm2 versus 59 ± 9*103 μm2 respectively, Figure 2A), which was also not affected 144.

(8) Adventitial Mast Cells Induce Plaque Destabilization. by cromolyn treatment during challenge of the animals (control: 54 ± 7*103 μm2 versus DNP-challenged: 66 ± 11*103 μm2). Medial surface area was slightly increased in the DNP-challenged mice (42 ± 4*103 μm2 versus 34 ± 2*103 μm2 in controls, p=0.04), which did not occur after cromolyn treatment (DNP-challenged: 36 ± 3*103 μm2 versus controls: 35 ± 3*103 μm2). A. B. Plaque size ( Mm2 ). 100000 80000 60000 40000 20000 0. DNP P<0.05. 8.0 6.0 4.0 2.0 0.0. Control. DNP. D. 100. P<0.001. P<0.001. E amount of neutrophils. P<0.05. 2. MC/mm tissue. 10.0. % activated mast cells. Control. C. 80 60 40 20. 125 100 75 50 25 0. 0. Control. DNP. Control. DNP. Figure 2 Mast cell content in the adventitia of atherosclerotic carotid artery lesions. (A) Intimal surface area of the locally challenged animals (black bars) and control and DNP-challenged animals which also received the mast cell stabilizer cromolyn (white bars). (B) Toluidin Blue staining of a resting (left panel) and an activated (degranulating, right panel) mast cell (indicated by arrows) in the adventitia of an atherosclerotic lesion (1000x). (C) Total adventitial mast cell content in control and DNP treated animals (black bars) and cromolyn treated control and DNP-challenged mice (white bars). (D) Adventitial mast cell degranulation in control and DNP-challenged mice, which was found to be signiicantly increased in the latter group. Cromolyn treatment normalized the levels of mast cell activation in the DNP-challenged animals. (E) Adventitial neutrophil levels did not differ between the groups and were not affected by cromolyn treatment.. Resting and activated mast cells in the adventitia of the lesions were detected by toluidin blue (Figure 2B). Alcian blue/saphranin O staining revealed that the majority of the adventitial mast cells were connective tissue-type mast cells (98.6%)36. Both the number of adventitial mast cells in DNP-challenged mice (7.5 ± 1.4 versus 4.8 ± 0.7 MC/mm2 adventitial tissue in the control mice, p<0.05, Figure 2C) and the percentage of degranulated adventitial mast cells were signiicantly increased (74.7 ± 3.9% versus 44.6 ± 5.7% in control animals, p<0.001, Figure 2D) at three days after perivascular challenge. Cromolyn treatment completely abolished the DNP-induced mast cell recruitment (3.1 ± 0.5 MC/mm2 adventitial tissue, p<0.01 compared to DNP-challenged mice that had not received cromolyn, Figure 2C), while also the amount of activated mast cells returned to basal levels (32.4 ± 4.9% in controls and 30.4 ± 7.4% in DNP mice that received cromolyn, Figure 2D). In the adventitia of these lesions, the total number of neutrophils did not differ between any of the groups (Figure 2E, P=NS), indicating that cromolyn did not affect neutrophil iniltration. Strikingly, further analysis of the plaque morphology revealed massive intraplaque hemorrhages, characterized by the presence of intimal erythrocytes, in 7 of 26 plaques of DNP-challenged animals (Figures 3A and F), while we observed no such phenomena in controls (Figure 3B, 0 of 27, p=0.004). CD31 positive microvessels 145.

(9) Chapter 9. were detected, generally in the close proximity of hemorrhages (Figure 3C). A Perl’s iron staining conirmed these indings (6 of 26 compared to 0 of 27 for control mice, Figures 3D, E and F; p=0.01). Iron staining was found to correlate with the presence of intraplaque hemorrhage (p<0.0001), co-localized with ceroid-rich regions (data not shown) and was conined mostly to the central atheroma. Of 6 iron positive lesions, 5 arteries revealed iron staining also in the media (p=0.02) and enhanced medial thickening. Importantly, none of the cromolyn treated DNP-challenged mice showed intraplaque hemorrhage or iron staining (p=0.01 compared to DNP-challenged animals that had not received cromolyn, Figure 3F). C. B. A. F - cromolyn D. E. IPH. Treatment Control DNP. +. 0 7. 27 19. Total. 21 20. Total. 27 26. Iron. Treatment Control. 27. Total. 0. DNP. 6. 20. 26. Treatment Control DNP. +. 21 20. Total. +. 27. + cromolyn Treatment Control DNP. IPH +. 1 0. 22 20. Iron. 1 0. 22 20. Figure 3: Increased incidence of intraplaque hemorrhage in DNP-challenged animals. (A,B) Hematoxilin/ Eosin staining of plaques from DNP (A) and vehicle control (B) challenged mice, demonstrating intraplaque hemorrhages only in the irst lesion (100x). In (A), a high power magniication of an intraplaque hemorrhage shows erythrocytes extravasated in the intima of a DNP-challenged mouse (400x). (C) CD31 staining of a microvessel present in a DNP-challenged lesion (1000x). (D,E) Perl´s iron staining of a DNP (D) and a vehicle control challenged artery (E), revealing large areas with iron deposits only in the DNP-challenged plaque (100x). (F) Quantiication of the number of plaques containing intraplaque hemorrhages (IPH) and iron deposits in vehicle and DNP-challenged mice, establishing a strongly increased frequency of hemorrhages after DNP challenge (**p=0.004), which was conirmed by Perl’s iron staining (*p=0.01). Cromolyn treatment prevented the increase in intraplaque hemorrhage and iron deposites in DNP-challenged animals (*p=0.01 compared to DNP-challenged mice).. In vivo apoptosis As mast cell degranulation was reported to promote apoptosis of vSMCs and EC12, 37, 38 , we stained sections for apoptotic cells by TUNEL staining (Figure 4A). Indeed, we observed a signiicant increase in TUNEL positive area in DNP-challenged lesions (3.3 ± 0.5% TUNEL stained area compared to 0.6 ± 0.2% in control animals, p=0.002). While the iron negative lesions of the DNP-challenged animals displayed enhanced levels of TUNEL positive area (2.6 ± 0.6% TUNEL positive area, p=0.02 compared to controls), the degree of apoptosis was even more pronounced in the iron positive sections (4.8 ± 0.6% TUNEL positive area, p=0.01, Figure 4B). These data were conirmed by scoring of the TUNEL positive nuclei in the plaque (6.1 ± 2.0% versus 2.1 ± 0.6% in the controls, p=0.04, Figure 4C). The majority of the apoptotic cells was located in the central atheroma rather than in the SMC rich lesion cap (p=0.04, Figure 4C), suggesting that adventitial mast cell degranulation preferentially induces macrophage apoptosis. Strikingly, no differences were observed between controls and DNP-challenged mice that had been treated with cromolyn (1.5 ± 0.7% versus 2.4 ± 0.9% TUNEL stained area, respectively, p=0.4), suggesting that cromolyn prevented the adventitial mast cell induced macrophage apoptosis. 146.

(10) Adventitial Mast Cells Induce Plaque Destabilization. Mast cell releasate induces macrophage apoptosis Releasates of MC/9 cells and of mast cell freshly isolated from the peritoneal cavity (peritoneal mast cells, PMCs) were analyzed after stimulation with compound 48/80 for their composition. MC/9 releasate was previously shown to contain histamine26 and was demonstrated to have β-hexosaminidase activity (1.3% of total release) as well as to contain tryptase (1.2% of total release). PMC releasate also showed β-hexosaminidase activity (16% of total release) and contained chymase, tryptase, VEGF and histamine (7.8%, 3.5%, 18.7% and 5.5% of total release, respectively). A. $.0. #ONTROL B. C 10. *. 6. *. 4 2 0. % apoptotic cells. % apoptotic area. 8. 7.5 5 2.5 0. Control. Iron -. Iron +. DNP. *. *. Intima. Core. Control. Cap. DNP. Figure 4: Increased apoptosis in lesions of DNP-challenged mice. (A) TUNEL staining of a vehicle (left panel) and a DNP (right panel) challenged artery; arrows indicate TUNEL positive nuclei in brown (200x). (B) Relative TUNEL positive intimal area of control, iron negative and iron positive DNP-challenged plaques, which both showed increased apoptosis in DNP-challenged plaques. (C) The percentage of TUNEL positive nuclei in the complete intima of vehicle controls is lower than that of DNP-challenged animals. The percentage of TUNEL positive nuclei was signiicantly increased in the central core of DNP-challenged lesions, while no signiicant difference was found in the percentage of TUNEL positive nuclei in the cap region of control and DNP-challenged plaques. *p<0.05 compared to the control.. Supernatant from MC/9 mast cells induced apoptosis of RAW 264.7 macrophages by up to 5-fold (21.8 ± 0.7% of apoptotic cells versus 4.4 ± 0.3% for control medium, Figure 5A). To pinpoint the culprit mast cell constituent, we assessed the effect of tryptase (leupeptin), chymase (SBTI) inhibitors as well as of histamine receptor antagonists on mast cell-induced apoptosis. SBTI, leupeptin and the H1receptor antagonist triprolidine were able to inhibit mast cell-induced macrophage apoptosis, while the H2- and H3-receptor antagonists cimetidine and thioperamide were ineffective (Figure 5B). None of the inhibitors affected endogenous apoptosis or H2O2 induced apoptosis of RAW 264.7 cells, thus excluding that the used inhibitors are proor anti-apoptotic by themselves (data not shown). As both the protease inhibitors and the H1-receptor antagonist completely inhibited macrophage apoptosis, we veriied whether histamine acts synergistically on tryptase-induced apoptosis or vice versa. Incubation with histamine for 16 hours strongly increased macrophage apoptosis (9-fold, p<0.01), while subsequent post-treatment with tryptase (6 hours) led to an additive (1.8-fold) increase in macrophage apoptosis compared to treatment with 147.

(11) Chapter 9. histamine only. Tryptase treatment for 6 hours appeared to be ineffective. Conversely, priming of macrophages with tryptase for 16 hours slightly enhanced RAW 264.7 cell apoptosis (13 ± 3% compared to 4 ± 2% for untreated cells, p<0.05), but did not sensitize macrophages for histamine induced apoptosis (data not shown). In analogy to the MC/9 experiments, the supernatant of PMCs and that of primary cultured bone marrow derived mast cells (BMMCs), both degranulated with compound 48/80, induced peritoneal macrophage apoptosis (Figure 5B). In agreement with previous studies12, 13, supernatant of degranulated MC/9 cells induced vSMC apoptosis after 48 hours (data not shown), although vSMC appeared to be less susceptible to mast cell induced apoptosis than macrophages. Furthermore, we determined whether mast cell supernatant from MC/9 cells, PMCs and BMMCs, degranulated with compound 48/80, was able to induce endothelial cell proliferation. Indeed, murine endothelial H5V cells showed an increase in proliferation up to 200% when exposed to supernatant of either the activated mast cell line or the primary cultured mast cells (p<0.05, Figure 5C). A. B 30. 10. control MC/9. 5x. D. ##. ##. 30. ** *. 20 10 0. Tryptase -. Histamine. +. + +. 20 10. #. 0 control MC/9 H1RA H2RA H3RA. F 50. 600. *. 40 30. *. *. 20. ***. 450 300. *. *. 150. 10 0. + -. *. 30. control 48/80 MC/9 SBTI leupeptin. E 40. % apoptotic cells. 10. 0. 50x. **. 20. % apoptotic cells. *. 3. 20. 40. dpm (*10 ). % apoptotic cells. **. % peritoneal macrophage apoptosis. % apoptotic cells. 30. 0. C. Control MC/9. PMC BMMC. 0. Control MC/9 PMC BMMC. Figure 5: In vitro apoptosis of macrophages induced by mast cell degranulation. Supernatant from degranulated MC/9 mast cells induced apoptosis of RAW 264.7 macrophages in a dose dependent fashion (5x and 50 x dilution of mast cell supernatant (*p<0.05, **p≤0.01 compared to DMEM control, A). MC/9 mast cell induced apoptosis of RAW 264.7 cells (**P≤0.01 compared to DMEM control) was inhibited by the chymase inhibitor SBTI and by the tryptase inhibitor leupeptin (##p≤0.01 compared to MC/9 supernatant, B). Compound 48/80, used to degranulate the MC/9 mast cells, did not exert any effect on macrophage apoptosis. Mast cell induced apoptosis was completely abolished by the H1-receptor antagonist triprolidine (#p<0.05 compared to the mast cell supernatant induced apoptosis), but not by the H2-receptor antagonist cimetidine and the H3-receptor antagonist thioperamide (C). Incubation of RAW 264.7 cells with 100 μM of histamine induced macrophage apoptosis, which was even enhanced after 6 hour pre-incubation with tryptase, while tryptase itself did not induce apoptosis after 6 hours (D). (E) Supernatant from compound 48/80 activated MC/9 cells, peritoneal mast cells (PMC) and bone marrow derived mast cells (BMMC) induced apoptosis of freshly isolated peritoneal macrophages (*p<0.05). (F) Also, supernatant from MC/9, PMC and BMMC cells was able to induce proliferation of H5V endothelial cells (*p<0.05, ***p<0.0001).. Cromolyn effects on leukocytes To determine whether cromolyn was mast cell speciic, we systemically challenged C57Bl6 mice and analyzed effects of cromolyn on peritoneal cell composition. Cromolyn treatment did not affect relative neutrophil (Figure 6A), monocyte (Figure 6B) or Tcell content (Figure 6C). However, the number of peritoneal CD117+ mast cells was signiicantly reduced by treatment with cromolyn (p<0.001, Figure 6D). Similar results 148.

(12) Adventitial Mast Cells Induce Plaque Destabilization. were obtained after cromolyn treatment of locally challenged C57Bl/6 mice (Supplemental Figure 1, page 156). In line with these data, after perivascular DNP challenge of mast cell deicient Kit(W-sh/W-sh) mice32, cromolyn did not affect neutrophil and monocyte levels in the peritoneal cavity (Supplemental Figure 2, page 156). Myeloperoxidase (MPO) activity of peritoneal neutrophils also remained unaltered by cromolyn treatment after systemic (Figure 6E) and local challenge (Figure 6F) in C57Bl/6 mice and did not differ in cromolyn treated Kit(W-sh/W-sh) mice after perivascular challenge (Figure 6G). Analysis of DNFB sensitized skin segments of C57Bl/6 mice revealed that cromolyn treatment did not affect the percentage of neutrophils and eosinophils. The percentage of mast cells was signiicantly enhanced in these mice after DNP challenge (p<0.01), an effect that was reduced by cromolyn administration (Supplemental Figure 3A, page 156). In Kit(W-sh/W-sh) mice, cromolyn did not alter the levels of skin neutrophils and eosinophils after DNFB sensitization, while mast cells were as expected undetectable in these mice (Supplemental Figure 3B, page 156). B. 9. 6. 3. 0. C. -ONOCYTES. 60. 45. 30. 15. DNP. Control. 14. 7. DNP. Control. DNP. 0.12. 0.12. 0.12. 0.09. 0.09. 0.09. 0.06. 0.06. 0.06. 0.03. 0.03. 0.03. Control. DNP. -AST#ELLS. 21. 14. 7. 0. Control. 0. 0. 0. 28. DNP. G. F. E. MPO (O.D.). 21. 0. 0. Control. D. 4 #ELLS. 28. CD117 + Mast Cells (% of total). .EUTROPHILS. CD3 + T-Cells (% of total). 12. CD11b+ Monocytes (% of total). GR1 + Neutrophils (% of total). A. Control. DNP. DNP. DNP + cromolyn. Figure 6: No side-effects of cromolyn on peritoneal cells and neutrophil activation. Cromolyn (white bars) did not affect neutrophil (A), monocyte (B) and T-cell levels (C) in the peritoneal cavity of systemically DNP challenged C57Bl6 mice, while mast cell levels were signiicantly reduced after cromolyn treatment (D,**p<0.001) compared to control and DNP mice without cromolyn treatment (black bars). Myeloperoxidase (MPO) activity in peritoneal neutrophils was not affected by cromolyn treatment after systemic (E) or local (F) challenge in C57Bl6 mice or in Kit(W-sh/W-sh) mice (G).. After systemic challenge in C57Bl/6 mice, serum levels of IL-2, IFNγ, IL-4 and TNFα were not signiicantly different between control and DNP challenged animals, although a trend towards reduced IL-4 levels was observed (p=0.07 compared to control mice, Supplemental Figure 4, page 157). As already mentioned above, we cannot exclude that systemic inlammatory responses after systemic mast cell activation may indirectly have inluenced atherogenesis. Therefore we next focused on mast cell activation locally at the lesion site. As aniticipated, after local challenge serum IFNγ remained undetectable, while serum IL-2, IL-4 and TNFα did not differ between the groups (Supplemental Figure 5, page 157). Similar results were obtained in Kit(W-sh/Wsh ) mice (Supplemental Figure 6, page 157). Microvascular leakage in vivo Apart from promoting macrophage apoptosis, mast cells have been suggested to induce vascular leakage39, 40. We indeed observed that after intradermal injection of 5*105 activated MC/9 cells in mice, vascular leakage as judged by Evans Blue spot size was signiicantly enhanced compared to PBS and was demonstrated to only be inhibited 149.

(13) Chapter 9. by the H1-receptor antagonist triprolidine (Supplemental Figure 7, page 158, p<0.001). More importantly, carotid artery lesions, perivascularly challenged with DNP and injected with Rhodamine 6G to label circulating leukocytes, contained a higher number of Rhodamine-positive cells than controls (p=0.0002, Figure 7A) at three days after challenge. Whereas in control mice the sparse Rhodamine-positive leukocytes were mainly found at the plaque surface, in the DNP-challenged mice a considerable portion of the Rhodamine-positive cells was detected in the central atheroma near the elastic lamina, suggesting that the latter had in part migrated through mast cell permeabilized microvessels inside the plaque. A Rhodamine positive leukocytes. 15. **. 10. 5. #ONTROL. $.0. 0. DNP. 60. 40. ** Leukocyte adhesion (cells/field). B. Leukocyte adhesion (cells/field). Control. 45 30 15. *. 30 20 10. #. Control. 4 X C M R et 2 -R A N T E S +. an t i-. C. -V LA. N P D. an ti. +. +. C. +. nt r +. co. DNP. an ti. Control. ol. 0. an VLA ti4 C X + C M R et -R 2 A N T E S. #. 0. DNP. Activated Mast Cell Intraplaque Macrophage Apoptosis Enhanced Neo-Vessel Permeability Chymase Tryptase Histamine (H1 - R). Plaque Destabilization. Leukocyte/Erythrocyte Influx. Figure 7: Increased leukocyte adhesion by perivascular mast cell activation. (A) Inlux of newly recruited Rhodamine+ leukocytes into DNP-challenged atherosclerotic plaques is increased compared to the control plaques (**p=0.0002, left panel). The middle and right panels show representative pictures of Rhodamine+ leukocytes, depicted by the white arrows, in control and DNP-challenged animals. (B) In vivo perfusion of DNP-challenged atherosclerotic plaques resulted in increased adhesion of Rhodamine+ leukocytes (**p=0.0009, left panel). Ex vivo perfusion of the carotid arteries revealed an increased adhesion of Calcein-labeled MonoMac6 cells to DNPchallenged arteries, which could be blocked with anti-CXCR2 and anti-VLA-4 antibody treatment, but not with Met-RANTES (right panel, *p=0.02 compared to vehicle control, #P<0.05 compared to DNP challenged mice). (C) Suggested mechanism of adventitial mast cell degranulation on atherosclerotic lesions.. In vivo intravital microscopy/ex vivo perfusion To establish whether adventitial mast cell activation affects the adhesion of leukocytes to atherosclerotic plaques from the luminal side, circulating leukocytes were Rhodamine-6G labeled in vivo and adhesion of these cells to DNP and control challenged atherosclerotic lesion was monitored by intravital microscopy. The adhesion of labeled leukocytes to plaques was 2.5 fold increased after perivascular mast cell activation (49 150.

(14) Adventitial Mast Cells Induce Plaque Destabilization. ± 6 versus 19 ± 4 leukocytes/microscopic ield for DNP versus vehicle control plaques, respectively, p=0.0009, Figure 7B). Ex vivo perfusion studies on the adhesion of Calceinlabeled monocytic MonoMac-6 cells conirmed these indings (25 ± 6 cells/ield in DNP-challenged arteries versus 9 ± 2 cell/ield in control arteries, p=0.02, Figure 7B). Strikingly, antibody blockade of either the KC receptor CXCR2 or the β1 intergrin and VCAM-1 receptor VLA-4 on the MonoMac-6 cells completely inhibited the mast cellmediated monocyte adhesion to the atherosclerotic plaque, while blockade of CCR1, CCR3 and CCR5 with Met-RANTES and isotype control antibodies had no effect. None of the treatment modalities signiicantly reduced monocytic cell adhesion to control atherosclerotic plaques in the area of interest. Discussion Activated mast cells have been identiied to accumulate in the arterial adventitia during plaque progression and are abundantly present in the adventitia of vulnerable and ruptured lesions. To date it remains to be clariied whether these adventitial mast cells contribute to plaque progression and whether these cells are instrumental in plaque rupture. To address this key question, we recruited and activated mast cells in the adventitia of atherosclerotic lesions in ApoE-/- mice. We demonstrate that systemic mast cell activation during atherogenesis leads to increased plaque progression, while focal activation of mast cells in the adventitia of advanced plaques was established to enhance the incidence of intraplaque hemorrhage. Inhibition of mast cell degranulation by the mast cell stabilizer cromolyn prevented these pathophysiological events. Systemic mast cell activation was seen to aggravate spontaneous plaque progression in the brachiocephalic artery of ApoE-/- mice, an effect that was not observed after prior mast cell stabilization with cromolyn. More importantly, as most mast cells are present in type V/VI human atherosclerotic lesions, we have addressed the effect of focal mast cell activation on pre-existing collar-induced carotid artery plaques, which are more easily accessible for local intervention. These atherosclerotic plaques, formed proximal to the collar, were previously shown to be shear-stress induced and absolutely lipid-dependent, thus representing a valid model of true atherosclerosis24, 41. The DNP challenge led to a striking and acute increase in the incidence of intraplaque hemorrhage within three days after challenge. Also, iron deposits were observed in the media of these lesions, suggesting that culprit factors likely entered the intima after adventitial release and crossing of the media. Lesions with intraplaque hemorrhage tended to contain relatively more adventitial mast cells than those lacking hemorrhages, which is in line with the observation of Laine et al.19, that the adventitia of ruptured lesions in human coronary artery species contained increased levels of mast cells. Importantly, the total number of mast cells detected in the adventitia of mouse carotid artery plaques corresponded with that observed in adventitial tissue of human plaques18, 19, indicating that the mouse model offers a realistic representation of the human situation. Intraplaque hemorrhage seemed to co-localize with ceroidrich regions in close proximity to microvessels. Interestingly, Kolodgie et al.42 reported that intraplaque hemorrhage is a potent pro-atherogenic stimulus and risk factor in plaque destabilization, as it is accompanied by deposition of erythrocyte associated cholesterol and enlargement of the necrotic core of the atherosclerotic plaque. This concurs with indings of Kockx et al.43, that phagocytosis of accumulated erythrocytes by activated macrophages leads to ceroid production and further plaque expansion, which may promote the formation of rupture prone lesions. Treatment of mice with cromolyn during DNP challenge normalized the extent of mast cell degranulation in the adventitia, while preventing intraplaque hemorrhage. Importantly, cromolyn treatment was demonstrated to be mast cell speciic at the dosage used in our animal models and did not exhibit any side-effects on other cell types such as neutrophils and macrophages. These data imply that inhibition of perivascular mast cell degranulation 151.

(15) Chapter 9. may help to maintain plaque stability. Mast cells were reported to induce apoptosis of cardiomyocytes31, vascular smooth muscle cells12, 13, 38 and endothelial cells37 in vitro, which could translate in a reduced plaque stability44, 45. Indeed, we observed a highly signiicant increase of intimal apoptotic nuclei in the DNP-challenged mice. To our surprise, apoptosis was mainly localized in the central atheroma, implying that the majority of apoptotic cells are of macrophage rather than of vSMC origin. To date, mast cell degranulation has not been linked to macrophage apoptosis. Macrophage apoptosis may very well result in an enlarged necrotic core of the lesions and in the release of tissue-factor rich apoptotic micro-bodies46, thereby decreasing plaque stability and promoting thrombosis. Our in vitro indings concurred with the in vivo data, in that macrophages displayed a high susceptibility to mast cell induced apoptosis, while vascular smooth muscle cells appeared to be less sensitive. Protease inhibitors were able to prevent the mast cell induced macrophage apoptosis and an H1-receptor antagonist could completely blunt macrophage apoptosis. Both tryptase and chymase were suggested to be pro-apoptotic by themselves12, 13, 38, but also to potentiate the pro-apoptotic action of histamine47, which was conirmed by our in vitro data. DNP challenge substantially increased intimal inlux of erythrocytes. We demonstrate here that mast cell degranulation enhances microvascular leakage (Figure 7c). Circulating leukocytes were seen to extravasate possibly through mast cell-permeabilized microvessels in response to mast cell derived chemotactic stimuli, judged by the increased presence of rhodamine-labeled leukocytes in the DNP treated plaque. In vitro studies showed endothelial proliferation in response to mitogenic factors, possibly Vascular Endothelial Growth Factor (VEGF) secreted by mast cells48, which could result in enhanced outgrowth of microvessels, transforming the lesion into even more leakage-prone plaques. In neovascularized areas of human coronary atheromas, mast cells were demonstrated to colocalize with intraplaque microvessels49. Furthermore, mast cells containing basic Fibroblast Growth Factor (bFGF), a potent angiogenic factor, were located near microvessels in the intima and adventitia of human coronary artery lesions50, suggesting that mast cells might indeed play a role in neoangiogenesis, vascular leakage and plaque progression. Furthermore, we demonstrated that perivascular mast cell activation increased leukocyte and in particular monocyte adhesion to the proximal area of atherosclerotic plaques in a CXCR2- and VCAM-1-dependent manner. Murine mast cells have previously been demonstrated to release the CXCR2 ligand KC51 (or its human orthologue interleukin8 (IL-8)52, 53), suggesting that the arrest-triggering response directly originates from activated mast cells. This is also conceivable as tissue-derived chemokines, such as IL8, can be abluminally internalized and transcytosed to the luminal side of endothelial cells, where the chemokines can exert their effects54. Previous studies in ex vivo perfused arteries have revealed that monocyte arrest on early atherosclerotic endothelium is also mediated by VLA-4 and triggered by KC via CXCR255. In conjunction with our results, this indicated interesting mechanistic parallels in the contribution of these molecules to atherogenic recruitment at different stages, e.g. between initial arrest and into advanced plaques destabilized by mast cells. In conclusion, we are the irst to provide in vivo proof that mast cells contribute signiicantly to atherosclerotic plaque progression. Moreover, we show that perivascular mast cells can promote macrophage apoptosis, increase leukocyte inlux and enhance microvascular leakage in pre-existing atherosclerotic plaques, resulting in a sharply increased risk of intraplaque hemorrhage and plaque destabilization. Thus, our present in vivo indings concur with previous in vitro indings16 and point to a signiicant role for activated mast cells in plaque stability and acute coronary syndromes. We propose that mast cell stabilization can be an effective new therapeutic entry in the prevention of acute coronary syndromes. 152.

(16) Adventitial Mast Cells Induce Plaque Destabilization. Acknowledgments The authors would like to thank M. Bot from the Division of Biopharmaceutics of the Leiden/Amsterdam Center for Drug Research in Leiden and R. Eggers from the Netherlands Institute for Neurosciences in Amsterdam for technical assistance. This study was supported by grant 016.026.019 from the Netherlands Organization for Scientiic Research (I. B., S.C.A.d.J.) and grant 2003T201 from the Netherlands Heart Foundation (E.B.).. 153.

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(18) Adventitial Mast Cells Induce Plaque Destabilization 34. Walls AF, Suckling AJ, Rumsby MG. IgG subclass responses and immediate skin sensitivity in guineapigs with chronic relapsing experimental allergic encephalomyelitis. Int Arch Allergy Appl Immunol. 1987;84(2):109-115. 35. Zernecke A, Schober A, Bot I, et al. SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ Res. 2005;96(7):784-791. 36. Nakano T, Sonoda T, Hayashi C, et al. Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deicient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells. J Exp Med. 1985;162(3):1025-1043. 37. Latti S, Leskinen M, Shiota N, et al. Mast cell-mediated apoptosis of endothelial cells in vitro: a paracrine mechanism involving TNF-alpha-mediated down-regulation of bcl-2 expression. J Cell Physiol. 2003;195(1):130-138. 38. Leskinen MJ, Kovanen PT, Lindstedt KA. Regulation of smooth muscle cell growth, function and death in vitro by activated mast cells--a potential mechanism for the weakening and rupture of atherosclerotic plaques. Biochem Pharmacol. 2003;66(8):1493-1498. 39. Evans TW, Rogers DF, Aursudkij B, et al. Inlammatory mediators involved in antigen-induced airway microvascular leakage in guinea pigs. Am Rev Respir Dis. 988;138(2):395-399. 40. Grega GJ, Adamski SW. Effects of local mast cell degranulation on vascular permeability to macromolecules. Microcirc Endothelium Lymphatics. 1991;7(4-6):267-291. 41. Dekker RJ, van Thienen JV, Rohlena J, et al. Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am J Pathol. 2005;167(2):609-618. 42. Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003;349(24):2316-2325. 43. Kockx MM, Herman AG. Apoptosis in atherogenesis: implications for plaque destabilization. Eur Heart J. 1998;19 Suppl G:G23-28. 44. von der Thusen JH, van Vlijmen BJ, Hoeben RC, et al. Induction of atherosclerotic plaque rupture in apolipoprotein E-/- mice after adenovirus-mediated transfer of p53. Circulation. 2002;105(17):20642070. 45. Bennett MR. Apoptosis in the cardiovascular system. Heart. May 2002;87(5):480-487. 46. 46. Hutter R, Valdiviezo C, Sauter BV, et al. Caspase-3 and tissue factor expression in lipid-rich plaque macrophages: evidence for apoptosis as link between inlammation and atherothrombosis. Circulation. Apr 27 2004;109(16):2001-2008. 47. Hur J, Kang MK, Park JY, et al. Pro-apoptotic effect of high concentrations of histamine on human neutrophils. Int Immunopharmacol. 2003;3(10-11):1491-1502. 48. Boesiger J, Tsai M, Maurer M, et al. Mast cells can secrete vascular permeability factor/ vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of fc epsilon receptor I expression. J Exp Med. 1998;188(6):1135-1145. 49. Kaartinen M, Penttila A, Kovanen PT. Mast cells accompany microvessels in human coronary atheromas: implications for intimal neovascularization and hemorrhage. Atherosclerosis. 1996;123(1-2):123-131. 50. Lappalainen H, Laine P, Pentikainen MO, et al. Mast cells in neovascularized human coronary plaques store and secrete basic ibroblast growth factor, a potent angiogenic mediator. Arterioscler Thromb Vasc Biol. 2004;24(10):1880-1885. 51. Schramm R, Schaefer T, Menger MD, et al. Acute mast cell-dependent neutrophil recruitment in the skin is mediated by KC and LFA-1: inhibitory mechanisms of dexamethasone. J Leukoc Biol. 2002;72(6):11221132. 52. Moller A, Lippert U, Lessmann D, et al. Human mast cells produce IL-8. J Immunol. 1993;151(6):32613266. 53. Kelley J, Hemontolor G, Younis W, et al. Mast cell activation by lipoproteins. Methods Mol Biol. 2006;315:341-348. 54. Middleton J, Neil S, Wintle J, et al. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell. 1997;91(3):385-395. 55. Huo Y, Weber C, Forlow SB, et al. The chemokine KC, but not monocyte chemoattractant protein-1, triggers monocyte arrest on early atherosclerotic endothelium. J Clin Invest. 2001;108(9):1307-1314.. 155.

(19) Chapter 9. Supplemental Figures !. " .EUTROPHILS. -ASTCELLS. Mast Cells (% of total). 28. 2.1. 1.4. 21. 14. CD117. GR1 Neutrophils (% of total). 2.8. 0.7. 7. 0. 0. Control. DNP. Control. DNP. Supplemental Figure 1: Cromolyn did not affect peritoneal cell composition. Cromolyn treatment (white bars) did not affect neutrophil levels (a), while also the monocyte and T-cell percentages in the peritoneal cavity of perivascularly DNP challenged C57Bl6 mice remained unaffected (data not shown, P=NS). As expected, mast cell levels (b) were signiicantly reduced after cromolyn treatment (*P<0.01) compared to DNP challenged mice without cromolyn treatment (black bars).. !. GR1 Neutrophils (% of total). CD11b Monocytes (% of total). ". -ONOCYTES. 60. 45. 30. 15. .EUTROPHILS. 8. 6. 4. 2. 0. 0. DNP. DNP + cromolyn. DNP. DNP + cromolyn. Supplemental Figure 2 Cromolyn did not affect peritoneal cell composition. In mast cell deicient Kit(W-sh/W-sh) mice, cromolyn did not alter the percentage of monocytes (a) and neutrophils (b) after perivascular challenge. !. " 12. 10. % of cell total. % of cell total. 10. 12. neutrophil eosinophil mast cell. 8 6. 8 6. 4. 4. 2. 2. 0. neutrophil eosinophil mast cell. 0 Control. Control + cromolyn. DNP. DNP + cromolyn. DNP. DNP + cromolyn. Supplemental Figure 3 Cromolyn did not affect skin cell distribution. (a) Analysis of skin segments of C57Bl6 mice, which had been sensitized with DNFB, revealed that cromolyn did not affect neutrophil (black bars) and eosinophil numbers (grey bars, both as percentage of total cell count). The percentage of mast cells (white bars) was signiicantly enhanced in these mice after DNP challenge compared to control challenged mice (*P<0.01), which could be partly inhibited by cromolyn administration. (b) In Kit(W-sh/W-sh) mice, cromolyn did not alter the levels of skin neutrophils (black bars) and eosinophils (grey bars) after DNFB sensitization.. 156.

(20) Adventitial Mast Cells Induce Plaque Destabilization !. " 0.06. 0.2. - cromolyn + cromolyn. - cromolyn + cromolyn 0.15. IFNG (O.D.). IL-2 (O.D.). 0.045. 0.03. 0.015. 0.1. 0.05. 0. 0. Control. DNP. Control. DNP. $. # 320. 0.08. - cromolyn + cromolyn. TNFA (O.D.). IL-4 (pg/ml). 240. 160. - cromolyn + cromolyn. 0.06. 0.04. 0.02. 80. 0. 0. Control. Control. DNP. DNP. Supplemental Figure 4 Serum cytokine levels after systemic DNP challenge. After systemic challenge of C57Bl6 mice, serum levels of IL-2 (a), IFNγ(b), IL-4 (c) and TNFα (d) were not signiicantly different between control and DNP challenged animals (black bars) or after cromolyn administration (white bars), although a trend towards reduced IL-4 levels after DNP challenge was observed (P=0.07 compared to control mice). A. 250. 0.1. - cromolyn + cromolyn. - cromolyn + cromolyn. - cromolyn + cromolyn. 200. 0.09 0.06. 0.08. TNFA (O.D.). IL-4 (pg/ml). 0.12. IL-2 (O.D.). C. B 0.15. 150 100. 0. 0. Control. 0. Control. DNP. 0.04 0.02. 50. 0.03. 0.06. Control. DNP. DNP. 300. 0.075. 0.08. 240. 0.06. 0.06. 0.04. TNFA (O.D.). 0.1. IL-4 (pg/ml). IL-2 (O.D.). Supplemental Figure 5 Serum cytokine levels after local DNP challenge. After local challenge of C57Bl6 mice, serum levels of IL-2 (a), IL-4 (b) and TNFα (c) were not signiicantly different between control and DNP challenged animals (black bars) or after cromolyn administration (white bars). IFNγ could not be detected in serum of these mice.. 180 120. 0. 0. 0. DNP. DNP + cromolyn. 0.03 0.015. 60. 0.02. 0.045. DNP. DNP + cromolyn. DNP. DNP + cromolyn. Supplemental Figure 6 Serum cytokine levels in mast cell deicient mice. After local challenge of Kit(W-sh/W-sh) mice, serum levels of IL-2 (a), IL-4 (b) and TNFα (c) were not signiicantly different after cromolyn treatment. IFNγ was not detectable in serum of these mice. 157.

(21) Chapter 9. skin reaction (mm ). 75. *** 50. 25. 0 MC/9. total leukocyte number (*100). 15. *. 10. * 5. 0. Control. PBS MC/9 Histamine. *. 50. 25 #. 0. SBTI Leupeptin total neutrophil number. PBS. B. 75. 2. 2. skin reaction (mm ). A. PBS MC/9 H1RA H2RA H3RA Histamine. 45. * 30. *. 15. 0 Control. PBS MC/9 Histamine. C. 0"3. -#. Supplemental Figure 7 Microvascular leakage was enhanced by mast cell activation. (a) Evans Blue spots in the skin of C57Bl/6 mice (left panel). Evans Blue+ surface area of MC/9 injected skin was larger than that of PBS control injected skin (***P<0.001) and remained unaffected by mast cell protease inhibitors (middle panel). Only the H1-receptor antagonist triprolidine reversed the MC/9 induced vascular leakage (#P=0.02 compared to the MC/9 cells, right panel). (b) Leukocyte iniltration in the MC/9 injected skin was increased (*P=0.03, left panel). Increased neutrophil recruitment was measured in MC/9 injected skin (*P=0.03, right panel). (c) De novo recruitment of circulating Rhodamine-6G labeled leukocytes to the PBS injected skin (400x, left panel). Increased iniltration of Rhodamine-6G labeled leukocytes was observed in skin injected with MC/9 mast cells (400x, right panel).. 158.

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(23) 1. 2. 3. 4. 5. 6. 7. 8. 160. Division of Biopharmaceutics, Leiden Amsterdam Center for Drug Research, Leiden University, 2333CC, Leiden, the Netherlands Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, 2300 RC Leiden, the Netherlands. Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo, Norway Center for Heart Failure Research, University of Oslo, Norway Max Delbrück Center of Molecular Medicine, Berlin, Germany Section of Clinical Immunology and Infectious Diseases, Rikshospitalet University Hospital, Oslo, Norway Research Institute for Internal Medicine, Rikshospitalet University Hospital, University of Oslo, 0027, Oslo, Norway Experimental Vascular Pathology group,Department of Pathology, CARIM, Academic University Hospital Maastricht, the Netherlands.

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