Neuropeptide Y receptor 1 is expressed by B ... - Wiley Online Library

Acta Pædiatrica ISSN 0803-5253

REGULAR ARTICLE

Neuropeptide Y receptor 1 is expressed by B and T lymphocytes and mast cells in infantile haemangiomas Elysia M. S. Tan1, Max G. Blackwell1, Jonathan C. Dunne1, Reginald Marsh1,2, Swee T. Tan ([email protected])1,3,*, Tinte Itinteang1,* 1.Gillies McIndoe Research Institute, Wellington, New Zealand 2.University of Auckland, Auckland, New Zealand 3.Centre for the Study and Treatment of Vascular Birthmarks, Wellington Regional Plastic, Maxillofacial and Burns Unit, Hutt Hospital, Wellington, New Zealand

Keywords Angiogenesis, Infantile haemangioma, Neural crest, Neuropeptide Y, Receptors

ABSTRACT Aim: We investigated the expression of neuropeptide Y (NPY), NPY receptor 1 (NPYR1)

Correspondence Swee T. Tan, ONZM, MBBS, PhD, FRACS, Gillies McIndoe Research Institute, PO Box 7184, Newtown 6242, Wellington, New Zealand. Tel: +64-4-2820366 | Email: [email protected]

Methods: Immunohistochemical (IHC) staining was performed on proliferating IHs from

Received 8 June 2016; revised 19 October 2016; accepted 23 November 2016. DOI:10.1111/apa.13684 *Equal senior authors.

and NPY receptor 2 (NPYR2) in infantile haemangiomas (IHs). six patients aged 4–13 (mean 8.7) months and involuted IHs from six patients aged 5–59 (mean 18.7) years, for the expression of NPY, NPYR1 and NPYR2. Protein and messenger ribonucleic acid expression corresponding to these proteins was investigated by Western blotting and NanoString analysis, respectively. Results: IHC staining, Western blotting and NanoString analysis demonstrated the presence of NPYR1, but not NPYR2, within proliferating and involuted IHs. IHC staining showed NPYR1 was expressed by B and T lymphocytes expressing CD45 and mast cells expressing tryptase. IHC staining demonstrated the presence of NPY on the NPYR1+ cells, but it was not detected by Western blotting or NanoString analysis. Conclusion: NPYR1, but not NPYR2, was present in IHs. The localisation of NPYR1 to B and T lymphocytes and mast cells suggests its role in the biology of IHs. The demonstration of NPY on the NPYR1+ cells, without active transcription, suggests that NPY was not being produced within IHs.

INTRODUCTION Infantile haemangiomas (IHs) typically undergo rapid proliferation during infancy (proliferative phase) followed by slow spontaneous involution over one to five years of age (involuting phase) with fibro-fatty deposition that replaces the cellular elements, up to 10 years of age (involuted phase) (1–3). Neuropeptide Y (NPY), a sympathetic neurotransmitter secreted with catecholamines from sympathetic nerves (4,5), is released during nerve stimulation, such as during stress, exercise or ischaemia (4). NPY acts on five receptors (NPYR1–NPYR5) to mediate a number of activities (5). The vasoconstriction and proliferation of vascular smooth muscle and neuronal precursors are primarily mediated by NPY receptor 1 (NPYR1) and the promotion of angiogenesis by activating NPYR1 and NPY receptor 2 (NPYR2) (5). Although the sympathetic nerves are the major source of circulating NPY in vivo, the endothelium possesses its own

Key notes

Abbreviations DAB, 3,3-Diaminobenzidine; DTT, Dithiothreitol; IF, Immunofluorescent; IH, Infantile haemangioma; IHC, Immunohistochemical; NPY, Neuropeptide Y; NPYR1, Neuropeptide Y receptor 1; NPYR2, Neuropeptide Y receptor 2; RNA, Ribonucleic acid.

292

NPY autocrine system, which includes the receptors and the peptide itself (4). Neural crest-derived tumours, such as neuroblastomas, pheochromocytomas and Ewing’s sarcoma family of tumours, synthesise and release neural-specific peptides such as NPY (5). NPY regulates their growth directly by inducing tumour cell proliferation and indirectly by stimulating angiogenesis (5). In a study with xenografts of nude mice, treatment with NPY stimulated proliferation of neuroblastoma cells, via NPYR2 activation and thus enhanced tumour growth, but it inhibited Ewing’s sarcoma family of tumours cell growth by NPYR1-mediated

We investigated the expression of neuropeptide Y (NPY), NPY receptor 1 (NPYR1) and NPY receptor 2 (NPYR2) in proliferating and involuted infantile haemangiomas (IHs). NPYR1, but not NPYR2, was present in IHs and localised to B and T lymphocytes and mast cells suggesting its role in the biology of IHs. The demonstration of NPY on the NPYR1+ cells, without active transcription, suggests NPY was not being produced within IHs.

©2016 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 2017 106, pp. 292–297

Tan et al.

apoptosis (5). In both tumours, treatment with NPY led to an increase in tumour vascularisation. Angiotensin II has been shown to promote the release of NPY from cells derived from pheochromocytoma (6) and promote proliferation of IH-derived cells (7). These observations led us to hypothesise a role for NPY in IH vasculogenesis. This study investigated the presence of NPY, and its receptors NPYR1 and NPYR2, in both proliferating and involuted IHs.

MATERIALS AND METHODS Tissue samples Six proliferating and six involuted IH samples from 12 patients aged 4–13 (mean 8.7) months and 5–59 (mean 18.7) years, respectively, were used for this study, which was approved by the Central Regional Health and Disability Ethics Committee (ref. no. 13CEN130). The study was carried out at the Gillies McIndoe Research Institute in Wellington, New Zealand, from November 2015 to January 2016. Immunohistochemical staining Four-micrometre-thick formalin-fixed paraffin-embedded sections of six proliferating and six involuted IHs from 12 patients were used for immunohistochemical (IHC) staining. Antigen retrieval was performed using AR9961 (catalogue number) sodium citrate (Leica, Sydney, NSW, Australia) at 95°C for 15 minutes. All sections underwent single 3,3-diaminobenzidine (DAB) IHC staining for the primary antibodies REF355A-16 GLUT-1 (Cell Marque, Rocklin, CA, USA) at a concentration of 1:200; SAB1404138 NPY (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1:200; BML-SA642 NPYR1 (Enzo Life Sciences, New York, NY, USA) at a concentration of 1:500; and PA1-41576 NPYR2 (Thermo Fisher Scientific, Rockford, IL, USA) at a concentration of 1:500, with detection using the Bond Polymer Refine Detection kit (Leica). Primary antibodies M0701 CD45 (Dako, Glostrup, Denmark) at a concentration of 1:1500 and NCLMCTRYP-428 tryptase (Leica) at a concentration of 1:300 were also used for immunofluorescent (IF) IHC staining. To examine protein colocalisation, selected representative slides of each phase of IHs underwent IF IHC staining with the same primary antibodies at the same concentrations, but using an appropriate secondary antibody combination of VEDK2488 VectaFluor Excel anti-mouse 488 (Vector Laboratories, Burlingame, CA, USA) at the readyto-use concentration and A21207 donkey anti-rabbit Alexa 594 (Life Technologies, Foster City, CA, USA) at a concentration of 1:500 for combinations that included NPYR1. All antibodies were diluted in Bond primary antibody diluent (Leica). Dual DAB IHC staining was performed using the Bond Polymer Refine Red Detection kit (Leica). All DAB and IF IHC staining was performed on the Leica Bond Rx autostainer (Leica). The DAB and IF IHC-stained slides were mounted using Surgipath micromount mounting media (Leica) or Vectashield HardSet mounting medium with 40 ,6-diamidino-2-phenylindole

Neuropeptide Y receptor 1 in haemangiomas

(Vector Laboratories), respectively. The positive controls that were used were human breast cancer and normal breast tissue, for NPY and NPYR1, and NPYR2, respectively, and human tonsillar tissue for CD45 and tryptase. The omission of the primary antibody in an IH sample provided an appropriate negative control. Counting of NPYR1+ cells Cell counting was performed on DAB IHC-stained slides of six proliferating and six involuted IHs stained for NPYR1. NPYR1+ cells were counted in six fields of view at 409 magnification, with averages taken of the counts. NanoString gene analysis Six snap-frozen proliferating and six involuted IH samples from the same cohort of patients included in DAB IHC staining were used to isolate total RNA for NanoString nCounter Gene Expression Assay (NanoString Technologies, Washington, DC, USA). RNA was extracted from frozen tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and subjected to the NanoString nCounter Gene Expression Assay performed by New Zealand Genomics Ltd, Dunedin, New Zealand. Probes for the genes encoding NPY, NPYR1 and NPYR2 were NM_000905.2, NM_ 000909.4 and NPYR2 NM_000910.2, respectively, which were designed and synthesised by NanoString Technologies. Raw data were analysed by nSolver software (NanoString Technologies) using standard settings and were normalised against the housekeeping gene, GUSB, using the NM_000181.3 probe. Western blotting Snap-frozen proliferating (n = 3) and involuted (n = 3) IH tissues from the same cohort of patients included in DAB IHC staining were analysed by Western blotting. The diced tissue samples of around 10 mg of tissue per sample were washed three times in ice-cold 19 phosphate-buffered saline and then homogenised in 350 lL ice-cold RIPA buffer (Sigma-Aldrich) supplemented with 10 mM dithiothreitol (DTT) and 19 HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) using an Omni TH mechanical homogeniser (Omni International, Kennesaw, GA, USA). The samples were centrifuged at 17 000 9 g at 4°C for 20 minutes, and the supernatants were collected in fresh microcentrifuge tubes. Solubilised proteins were precipitated for 1 hour at 20°C using the ProteoExtract Protein Precipitation Kit (Merck Millipore, Billerica, MA, USA) and resuspended overnight at 4°C in 19 Laemmli buffer (Bio-Rad, Hercules, CA, USA) containing 10 mM DTT. In addition, samples for NPY Western blotting analysis were prepared by Dounce homogenisation of around 10 mg of tissue per sample in 30 lL IP lysis buffer (Thermo Fisher Scientific) containing 10 mM dithiothreitol (DTT) and 19 HALT protease and phosphatase inhibitor cocktail, without subsequent protein precipitation. Samples were prepared for sodium dodecyl sulphate– polyacrylamide gel electrophoresis by supplementing equal amounts of total protein with fresh 19 Laemmli buffer


293


Tan et al.

(Bio-Rad) containing 10 mM DTT (Bio-Rad; 40 lL final sample volume) and heating at 95°C for 10 minutes. Samples for NPY detection were supplemented with an equal volume of 29 Laemmli buffer (Bio-Rad) containing 10 mM DTT and heated at 95°C for 10 minutes. Samples were resolved using Bolt 4–12% Bis-Tris Plus gels (Life Technologies) for approximately 30 minutes at a constant voltage of 200 V and then transferred onto nitrocellulose membranes (Life Technologies) using an iBlot 2 (Life Technologies). The membranes were blocked immediately in 19 iBind Flex Solution (Life Technologies) at 4°C for 90 minutes and probed with appropriate primary and secondary antibodies using an iBind Flex device (Life Technologies). The following antibodies, indicated by their catalogue numbers, were used: primary antibodies SAB1404138 NPY (SigmaAldrich) at a concentration of 1:1000; sc-21992 NPYR1 (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:200, PA1-41576 NPYR2 (Thermo Fisher Scientific) at a concentration of 1:200 and ab8226 b-actin (Abcam, Cambridge, MA, USA) at a concentration of 1:2000; and secondary antibodies A16110 goat anti-rabbit HRP (Thermo Fisher Scientific) at a concentration of 1:10000, ab97120 donkey anti-goat HRP (Abcam) at a concentration of 1:5000 and A21239 Alexa Fluor 647 rabbit anti-mouse (Thermo Fisher Scientific) at a concentration of 1:2000. Clarity Western ECL substrate (Bio-Rad) was used to visualise HRP-immunoreactive protein bands. The ChemiDoc MP Imaging System and Image Lab 5.0 software (Bio-Rad) were used for image detection and analysis. Positive controls were synthetic 5017 NPY (SigmaAldrich), neuroblastoma cell line total protein extract for NPYR1 and breast tissue total protein extract for NPYR2. Image analysis DAB and IF IHC-stained slides were viewed and imaged using an Olympus BX53 light microscope fitted with an Olympus DP21 digital camera (Olympus, Tokyo, Japan) and an Olympus FV1200 confocal laser-scanning microscope and processed with cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus), respectively. Statistical analyses The statistical analysis was performed using Student’s t-test for independent samples from SPSS, version 22 (IBM Corp, New York, NY, USA), which included calculating for Levene’s test for equality of variances.

RESULTS Immunohistochemical staining All tissue samples were confirmed to be IHs with the expression of GLUT-1 (8) (data not shown). DAB IHC staining demonstrated the expression of NPYR1 on the endothelium of the microvessels (Fig. S1A) and cells in the interstitium (Fig. S1A) in proliferating and involuted (Fig. S1B) IHs. NPYR2 was not expressed in either proliferating (Fig. S1C) or involuted (Fig. S1D) IHs. DAB IHC staining also showed NPY immunoreactivity in both

294

the endothelium of the microvessels (Fig. S1E) and cells in the interstitium (Fig. S1E) in proliferating (Fig. S1E) and involuted (Fig. S1F) IHs. Positive staining was demonstrated in human breast cancer for NPYR1 (Fig. S2A), normal breast tissue for NPYR2 (Fig. S2B) and breast cancer for NPY (Fig. S2C) and tonsillar tissue for CD45 and tryptase (data not shown). Specificity of the secondary antibodies was demonstrated by the omission of the primary antibody in an IH sample (Fig. S2D). IF IHC staining demonstrated that NPYR1 (Fig. 1A and B, red) and NPY (Fig. 1A and B, green) were colocalised to the endothelium of the microvessels and the interstitial cells within proliferating (Fig. 1A) and involuted (Fig. 1B) IHs. IF IHC staining also demonstrated a subpopulation of cells coexpressing NPYR1 (Fig. 1C and D) and CD45 (Fig. 1C and D) and another subpopulation of cells coexpressing NPYR1 (Fig. 1E and F) and tryptase (Fig. 1E and F), in both proliferating (Fig. 1C and E) and involuted (Fig. 1D and F) IHs, respectively. Only a subpopulation of the CD45+ cells (Fig. 1C and D) coexpressed NPYR1 (Fig. 1C and D). Separated IF IHC stains for each marker are shown in Figure S3. Analysis of NPYR1+ cell counts The cell count averages for NPYR1+ cells in proliferating and involuted IHs are shown in Figure 2. There was no significant difference in the number of NPYR1+ cells in the proliferating and involuted IH groups (p < 0.05). NanoString gene analysis NPYR1 messenger RNA was present within both proliferating and involuted IHs (Fig. 3), but the amounts of messenger RNA between these two groups were not significantly different (p < 0.05). NPY and NPYR2 messenger RNA was not present within either group (data not shown). Western blotting Tissue lysates from three proliferating and three involuted IH samples from the IHC cohort were analysed by Western blotting. Equal protein loading across samples of around 30 lg per sample was confirmed using b-actin (Fig. S4A). A band at the expected size of 47 kDa for NPYR1 was identified in all proliferating and involuted IH samples (Fig. 4). NPYR2 was detected in the placenta positive control at around 50 kDa, which is around 7 kDa larger than the predicted size of the native protein and may indicate the presence of a post-translational modification. No corresponding bands were detected in any of the IH samples examined (Fig. S4B). Similarly, NPY was undetectable in all six IH samples and was also undetectable in two proliferating IH samples when around 300 lg of total protein per sample was analysed (Fig. S4C).

DISCUSSION This study demonstrated the expression of NPY and NPYR1, but not NPYR2, on both the endothelium of the microvessels and the cells in the interstitium of both


Tan et al.


A

B

C

D

E

F

Figure 1 Representative immunofluorescent immunohistochemical-stained sections of proliferating (A, C, E) and involuted (B, D, F) IHs showing coexpression of NPYR1 (A and B, red) with NPY (A and B, green). A subpopulation (arrowheads) of NPYR1+ cells (C and D, red) coexpressed CD45 (C and D, green), and another subpopulation (arrowheads) of NPYR1+ cells (E and F, red) coexpressed tryptase (E and F, green). Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue). Scale bar: 20 lm.

proliferating and involuted IHs. IHC staining demonstrated the expression of NPY, but this was not corroborated by Western blotting and NanoString analysis. The relatively few NPY+ cells may have possibly hindered detection by immunoblotting. The lack of NPY messenger RNA would suggest that NPY was produced elsewhere and recruited to the IH tissues from the circulation (9). Studies have demonstrated a crucial role for both NPY via NPYR1 in the maintenance of the haematopoietic stem

cell and the endothelial cell components of mice bone marrow (10). Cells of both lymphoid and myeloid lineages have been demonstrated within the interstitium of IHs (11,12) including a number of distinct interstitial cellular subpopulations: (i) phenotypic mast cells coexpressing tryptase and Nanog within proliferating IHs, indicating a primitive phenotype; (ii) M2-polarised macrophages, characterised by the expression of CD163 and DC-SIGN (12); and (iii) a further subpopulation that expresses the


295


Tan et al.

Figure 2 Bar graph showing the average total number of NPYR1+ cells within proliferating and involuted infantile haemangiomas (IHs). Results are presented as means and standard deviation. There was no statistically significant difference between the number of NPYR1+ cells within proliferating and involuted IHs.

Figure 4 Representative 1DE Western blot image showing detection of NPYR1 in both proliferating and involuted infantile haemangioma samples at the expected size of 47 kDa. b-Actin was used as a loading control and confirmed equivalent total protein load across all samples.

Figure 3 NPYR1 messenger RNA levels in six proliferating and six involuted infantile haemangioma (IH) tissues. Messenger RNA level was assessed using NanoString nCounter Gene Expression Assay with specific probes for NPYR1, NPY and NPYR2 genes. The results are presented as mean and standard deviation. There was no statistically significant difference in the amounts of NPYR1 messenger RNA between proliferating and involuted IHs.

pan-haematopoietic cell marker CD45, but not Nanog, CD163, DC-SIGN or tryptase (12). This study demonstrated the presence of NPYR1 and its ligand, NPY, on the mast cells and CD45+ cells in the interstitium of IHs. This finding suggests a putative role for NPYR1 activation by NPY, resulting in the promotion of microvascular proliferation characteristic of the proliferative phase of IHs, although conclusive evidence for this remains a topic of further investigation. NPY-deficient mice have been shown to exhibit a significantly reduced number of haematopoietic stem cells, as well as impaired regeneration in bone marrow due to apoptotic destruction of sympathetic nervous system fibres and/or endothelial cells (13). Taken together, this suggests that the reduction in NPYR1+ cells during involution demonstrated in this study may, in part, explain the observed increased apoptosis evident in involuted IHs (14).

296

The novel findings of this study of the presence of NPY and NPYR1 on the phenotypic mast cells within IHs suggest a role for the NPY ligand and the putative activation of NPYR1 in promoting cellular proliferation and/or activation of the haematopoietic interstitial component of IHs. This may, in turn, result in the putative secretion of cytokines that contribute to the proliferation of the vasculature in proliferating IHs, although this remains the topic of further investigation. This report highlights novel insights into the NPY/NPYR1 axis and the contribution of the haematopoietic cells in the promotion and/or maintenance of the niche that promotes vasculogenesis during proliferation of IHs.

CONCLUSION This study demonstrated the presence of NPYR1 on tryptase+ and CD45+ haematopoietic cells in IHs. This finding infers a potential role for NPYR1 and its ligand, NPY, in both the proliferation of the endothelium and the development and activation of the phenotypic mast cell population within IHs. However, this remains to be conclusively determined.

ACKNOWLEDGEMENTS We thank Ms Liz Jones of the Gillies McIndoe Research Institute for her assistance in IHC staining, Dr Andrea Mikulasova for processing the tissue samples for NanoString testing and Ms Alice M Chibnall for the analysis of the data.


Tan et al.

CONFLICT OF INTEREST The authors have no conflict of interests to declare.

FUNDING No external funding was received for this study.

References 1. Itinteang T, Withers AH, Davis PF, Tan ST. Biology of infantile hemangioma. Front Surg 2014; 1: 38. 2. Munden A, Butschek R, Tom W, et al. Prospective study of infantile haemangiomas: incidence, clinical characteristics and association with placental anomalies. Br J Dermatol 2014; 170: 907–13. 3. Tan CE, Itinteang T, Leadbitter P, et al. Low-dose propranolol regimen for infantile haemangioma. J Paediatr Child Health 2014; 51: 419–24. 4. Zukowska-Grojec Z, Karwatowska-Prokopczuk E, et al. Neuropeptide Ya novel angiogenic factor from the sympathetic nerves and endothelium. Circ Res 1998; 83: 187– 95. 5. Kitlinska J, Abe K, Kuo L, et al. Differential effects of neuropeptide Y on the growth and vascularization of neural crest–derived tumors. Cancer Res 2005; 65: 1719–28. 6. Grouzmann E, Werffeli-George P, Fathi M, et al. AngiotensinII mediates norepinephrine and neuropeptide-Y secretion in a human pheochromocytoma. J Clin Endocrinol Metab 1994; 79: 1852–6. 7. Itinteang T, Marsh R, Davis PF, et al. Angiotensin II causes cellular proliferation in infantile haemangioma via angiotensin II receptor 2 activation. J Clin Pathol 2015; 68: 346–50. 8. North PE, Waner M, Mizeracki A, et al. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol 2000; 31: 11–22. 9. Hauser G, Danchak M, Colvin M, et al. Circulating neuropeptide Y in humans: relation to changes in catecholamine levels and changes in hemodynamics. Neuropeptides 1996; 30: 159–65. 10. Park MH, Jin HK. Perspective: role of neuropeptide Y in the bone marrow hematopoietic stem cell microenvironment. BMB Rep 2015; 48: 645–6. 11. Tan EM, Itinteang T, Chudakova DA, et al. Characterisation of lymphocyte subpopulations in infantile haemangioma. J Clin Pathol 2015; 68: 812–8. 12. Tan EM, Chudakova DA, Davis PF, et al. Characterisation of subpopulations of myeloid cells in infantile haemangioma. J Clin Pathol 2015; 68: 571–4.


13. Park MH, Jin HK, Min WK, et al. Neuropeptide Y regulates the hematopoietic stem cell microenvironment and prevents nerve injury in the bone marrow. EMBO J 2015; 34: 1648–60. 14. Razon MJ, Krӓling BM, Mulliken JB, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation 1998; 5: 189–95.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Representative 3,3-diaminobenzidine immunohistochemical-stained sections of infantile haemangiomas (IHs) showing the expression of NPYR1 by the endothelium of the microvessels (arrows) and the interstitial cells (arrowheads) in proliferating (A, brown) and involuted (B, brown) IHs, but not NPYR2 in either proliferating (C) or involuted (D) IHs. NPY was expressed by the endothelium of the microvessels (arrows) and the interstitial cells (arrowheads) in proliferating (E, brown) and involuted (F, brown) IHs. Original magnification: 4009. Figure S2. 3,3-Diaminobenzidine immunohistochemicalstained images of positive control human samples using human breast cancer for NPYR1 (A, brown), normal breast tissues for NPYR2 (B, brown) and breast cancer for NPY (C, brown). The negative control (D) confirmed specificity of the secondary antibody on a section of infantile haemangioma tissue by omitting the primary antibody. Original magnification: 4009. Figure S3. Split immunofluorescent immunohistochemicalstained images of proliferating (A, B, E, F, I and J) and involuted (C, D, G, H, K, L) infantile haemangiomas (IHs) showing expression of NPYR1 (A, C, E, G, I and K, red), NPY (B and D, green), CD45 (F and H, green) and tryptase (J and L, green). Scale bars: 20 lm. Figure S4. Representative 1DE Western blot images of proliferating and involuted infantile haemangioma (IH) samples. Equal loading of total protein was confirmed by bactin detection (A). NPYR2 was not detected at the expected size of 43 kDa (B). NPY was undetectable in two proliferating IH samples after approximately 10-fold sample concentration. Synthetic NPY was used as the positive control (C).


297