The American Journal of Pathology, Vol. 189, No. 3, March 2019 ajp.amjpathol.org NEUROBIOLOGY Expression of Bone Morphogenetic Proteins in Multiple Sclerosis Lesions Carme Costa,*y Herena Eixarch,*y Elena Martínez-Sáez,yz Laura Calvo-Barreiro,*y Maite Calucho,*y Zoraida Castro,*y Arantxa Ortega-Aznar,yz Santiago Ramón y Cajal,yz Xavier Montalban,*yx and Carmen Espejo*y From the Neurology-Neuroimmunology Department,* Multiple Sclerosis Centre of Catalonia (Cemcat), Vall d’Hebron Research Institute (VHIR), Vall d’Hebron University Hospital, Barcelona, Spain; Autonomous University of Barcelona,y Barcelona, Spain; the Pathology Department,z Vall d’Hebron University Hospital, Autonomous University of Barcelona, Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain; and the Division of Neurology,x BARLO Multiple Sclerosis Centre, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada Accepted for publication November 2, 2018. Bone morphogenetic proteins (BMPs) are secreted proteins that belong to the transforming growth factor-b superfamily. In the adult brain, they modulate neurogenesis, favor astrogliogenesis, and Address correspondence to Carmen Espejo, Ph.D., Servei inhibit oligodendrogenesis. Because BMPs may be involved in the failure of remyelination in multiple de Neurologia- sclerosis (MS), we characterized the expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMP type II Neuroimmunologia, Centre receptor (BMPRII); and phosphorylated SMAD (pSMAD) 1/5/8 in lesions of MS and other demyelinating d’Esclerosi Múltiple de diseases. A total of 42 MS lesions, 12 acute ischemic lesions, 8 progressive multifocal leukoence- Catalunya, Vall d’Hebron phalopathy lesions, and 10 central nervous system areas from four nonneuropathological patients were Institut de Recerca, Hospital included. Lesions were histologically classified according to the inflammatory activity. The expression of Universitari Vall d’Hebron, BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified by immunostaining, and Ps Vall d’Hebron 119-129, colocalization studies were performed. In MS lesions, astrocytes, microglia/macrophages, and neurons Collserola Building, 08035 expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8. Oligodendrocytes expressed Barcelona, Spain. E-mail: BMP-2 and BMP-7 and pSMAD1/5/8. The percentage of cells that expressed BMPs, BMPRII, and carmen.espejo@vhir.org. pSMAD1/5/8 correlated with the inflammatory activity of MS lesions, and changes in the percentage of positive cells were more relevant in MS than in other white matteredamaging diseases. These data indicate that BMPs are increased in active MS lesions, suggesting a possible role in MS pathogenesis. (Am J Pathol 2019, 189: 665e676; https://doi.org/10.1016/j.ajpath.2018.11.007) Bone morphogenetic proteins (BMPs) are secreted factors lineage fate. In the CNS, BMPs favor astrogliogenesis as that are members of the transforming growth factor-b su- they simultaneously impede the differentiation and matu- perfamily. Approximately 20 BMPs have been described ration of oligodendrocyte progenitor cells (OPCs) into and classified into subgroups according to sequence simi- mature oligodendrocytes, thus disturbing myelination.6,7 larities and functions.1 BMP receptors are transmembrane serine/threonine kinase receptors that are classified into two subgroups: types I and II.2 As dimeric ligands, BMPs bind Supportedby the Fondo de Investigación Sanitaria (FIS) grant PI12/ to type II dimeric receptors and subsequently recruit type I 02144 (C.E.); Instituto de Salud Carlos III, Ministry of Economy, Industry and Competitivity, Spain; the Fundación Salud 2000eMerk Serono, Spain dimeric receptors to form a heterotetrameric receptor com- grant SERONO-2013-04 (C.E.); the Red Española de Esclerosis Múltiple plex that signals through SMAD1/5/8.3 Phosphorylated grant RD12/0032 (X.M.), sponsored by the FIS; and the Ajuts per donar SMAD (pSMAD) 1/5/8 translocates into the nucleus and Suport als Grups de Recerca de Catalunya grant 2014 SGR 1082 (X.M.), regulates the transcription of BMP-dependent genes.4 Their sponsored by the Agència de Gestió d’Ajuts Universitaris i de Recerca, role in the developing central nervous system (CNS) has Generalitat de Catalunya, Spain. C.C. and H.E. contributed equally to this work. been well studied,5 and it is known that BMPs are involved Disclosures: X.M. reports personal fees from Actelion, Bayer, Biogen, in many physiological processes in adults. BMPs are mor- Celgene, Hoffmann-La Roche, Merck, Novartis, Oryzon Genomics, Sanofi- phogens that are mainly involved in the regulation of cell Genzyme, and Teva Pharmaceutical, not relevant to this work. Copyright ª 2019 American Society for Investigative Pathology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0). https://doi.org/10.1016/j.ajpath.2018.11.007 Costa et al Table 1 Characteristics of MS Brain Blocks Age, Postmortem Patient no. Sex years delay, hours MS type CHIAs, n CLIAs, n CIAs, n Cause of death EM1 F 62 17 RRMS 0 0 1 Pulmonary thromboembolism EM2 M 45 10 RRMS 0 3 3 Acute myocardial infarct EM3 F 50 20 RRMS 4 2 2 Bronchopneumonia EM4 F 46 <24 RRMS 4 1 1 Septic endocarditis (Escherichia coli) EM5 M 47 <24 RRMS 17 3 1 Pulmonary thromboembolism F, female; M, male; CHIA, chronic MS lesion with high inflammatory activity; CIA, chronic inactive MS lesion; CLIA, chronic MS lesion with low inflammatory activity; MS, multiple sclerosis; RRMS, relapsing-remitting MS. Similarly, BMPs are up-regulated when demyelination oc- not only) caused by the local immune response, including curs, favoring the differentiation of astrocytes over oligo- demyelination, axonal loss, and gliosis, clearly contribute to dendrocytes, probably hindering the repair of the damaged the neurodegeneration that leads to a chronic disability.11 tissue.8 BMPs are essential to maintain the undifferentiated Although there is evidence of extensive remyelination state of neural stem cells within the adult CNS. In fact, a even in lesions of long-diseased MS patients,12,13 proper precise regulation of BMP signaling is necessary to main- remyelination does not take place in a significant proportion tain the balance between quiescence/self-renewal and pro- of MS lesions. Data support that OPC migration and dif- liferation/differentiation processes to ensure appropriate ferentiation are impaired in MS,14 and that premyelinating neurogenesis throughout adulthood.9 oligodendrocytes are found in chronic lesions, although they Because BMPs are important in demyelination, oligo- are not capable of remyelinating.15 The cause of remyeli- dendrocyte differentiation, and maturation, as well as in the nation failure in MS is far from being understood; never- control of the neural stem cell pool, their signaling pathway theless, several hypotheses have been proposed, including may represent a putative therapeutic target for multiple the presence of inhibitory molecules in the microenviron- sclerosis (MS), a chronic, demyelinating, immune-mediated ment of the lesion that would impede the recruitment and disease of the CNS.10 Alterations in the CNS mainly (but differentiation of OPCs that promote tissue repair in the Table 2 Antibodies and Conditions Used for Immunohistochemistry Target Antibody Source (catalog number) Antigen retrieval Dilution Primary antibodies BMP-2 Goat antieBMP-2 (N-14) Santa Cruz Biotechnology (sc-6895) Citrate (pH 6) 1:100 BMP-4 Mouse antieBMP-4 (6B7) Cell Signaling Technologies, 1:100 Danvers, MA (number 4680) BMP-5 Goat antieBMP-5 (N-19) Santa Cruz Biotechnology (sc-7405) Citrate (pH 6) 1:100 BMP-7 Goat antieBMP-7 (L-19) Santa Cruz Biotechnology (sc-9305) 1:100 BMPRII Goat anti-BMPRII (T-18) Santa Cruz Biotechnology (sc-5683) TE (pH 9) 1:100 pSMAD1/5/8 Goat anti-pSMAD1/5/8 Santa Cruz Biotechnology (sc-12353) TE (pH 9) 1:100 (Ser463/Ser465) Neurons Mouse anti-NeuN Millipore, Burlington, MA (MAB377) 1:200 Rabbit anti-NeuN Sigma (HPA030790) 1:100 Oligodendrocytes Rabbit anti-Olig2 Millipore (AB9610) 1:100 Astrocytes Rabbit anti-GFAP DakoCytomation, Glostrup, 1:500 Denmark (Z0334) Mouse anti-GFAP Cy3 Sigma (C9205) 1:500 Microglia/macrophages Mouse anti-CD68 DakoCytomation (M0876) 1:400 Rabbit anti-CD68 Sigma (HPA048982) 1:100 Secondary antibodies Rabbit anti-goat HRP DakoCytomation (P0449) 1:200 Goat anti-mouse DakoCytomation (P0447) 1:200 HRP Goat anti-mouse Alexa 568 Life Technologies, Carlsbad, 1:200 CA (A11031) Goat anti-rabbit Alexa 568 Life Technologies (A11011) 1:200 Goat anti-mouse Alexa 488 Life Technologies (A11029) 1:200 Donkey anti-goat 488 Life Technologies (A11055) 1:200 BMP, bone morphogenetic protein; BMPRII, BMP type II receptor; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; NeuN, neuronal nuclei; Olig2, oligodendrocyte transcription factor 2; pSMAD, phosphorylated SMAD. 666 ajp.amjpathol.org - The American Journal of Pathology BMPs in MS Lesions Figure 1 Characterization of bone morphoge- netic proteins (BMPs), BMP type II receptor (BMPRII), and phosphorylated SMAD (pSMAD) 1/ 5/8 in multiple sclerosis (MS) lesions. Micrographs of chronic with high inflammatory activity MS le- sions showing the expression of BMP-2, BMP-4, BMP-5, and BMP-7, pSMAD1/5/8, and BMPRII (green) in different cell types (red): neurons (NeuN), oligodendrocytes (Olig2), astrocytes [glial fibrillary acidic protein (GFAP)], and macrophages (CD68). All images, except NeuN staining, were taken in the white matter at the margins of MS lesions where major inflammatory activity is found. Neurons were detected in the gray matter adjacent to the lesion. Arrows indicate colocali- zation of immunofluorescence staining. Scale bar Z 100 mm (all images). Original magnifica- tion, 40 (all images). NeuN, neuronal nuclei; Olig2, oligodendrocyte transcription factor 2. damaged areas.16 We, therefore, hypothesize that BMPs University Hospital, were retrospectively selected: 42 may contribute to the pathogenesis of MS. In this study, we demyelinating lesions from five relapsing-remitting MS describe the expression of BMPs in chronic MS lesions and patients (Table 1), 12 lesions from eight patients with acute determine whether their expression is relevant in MS cerebral infarct (AI), 8 lesions from four patients with pro- compared with other demyelinating diseases. gressive multifocal leukoencephalopathy (PML), and 10 normal-appearing white matter and normal-appearing gray matter (NAGM) areas from four nonneurologic control Materials and Methods (NNC) patients. The autopsy delay was <24 hours, and all Tissue Samples samples were fixed in 10% buffered formalin for 4 weeks and embedded in paraffin. Seventy-two CNS areas from 21 patients with neuropatho- The study was approved by the Clinical Research Ethics logical postmortem study, performed between 1992 and Committee of Vall d’Hebron University Hospital [PR(AG) 2009 at the Pathology Department, Vall d’Hebron 304/2012]. The American Journal of Pathology - ajp.amjpathol.org 667 Costa et al 668 ajp.amjpathol.org - The American Journal of Pathology BMPs in MS Lesions Histopathological Characterization of Demyelinated that underwent the same protocol but that were only Lesions and Classification of MS Lesions immunostained with the appropriate secondary antibodies were included. Formalin-fixed, paraffin-embedded blocks were cut into Double-immunofluorescence staining for BMP-2, BMP-4, serial sections (4 mm thick) and stained with hematoxylin BMP-5, and BMP-7; BMP type II receptor (BMPRII); and eosin and Klüver-Barrera for inflammation and demy- pSMAD1/5/8; and different cell types, including oligoden- elination assessment, respectively. MS lesions were subse- drocytes, neurons, astrocytes, and microglia/macrophages, was quently classified into chronic with high inflammatory performed. After deparaffinization, nonspecific protein binding activity (CHIA; n Z 25), chronic with low inflammatory was blocked with 2% bovine serum albumin and 0.3% Triton activity (CLIA; n Z 9), and chronic inactive (CIA; X-100 (Sigma) in phosphate-buffered saline solution. Antigen n Z 8).17 Briefly, CHIA lesions were hypercellular, with retrieval was performed in citrate buffer (pH 6) or TE buffer perivascular and lesion border inflammatory infiltration and (pH 9) when needed (Table 2). Sections were incubated active demyelination. CLIA lesions showed hypocellularity overnight at 4 C with primary antibodies (Table 2). Appro- with scarce perivascular inflammatory infiltration and no priate secondary antibodies were incubated for 1 hour at room active demyelination. CIA lesions showed neither inflam- temperature (Table 2). Samples incubated with the secondary mation nor active demyelination. AI lesions included the antibody were included as negative controls. acute ischemic core with pale neuropil and acute ischemic changes in neurons (piknotic nuclei, eosinophilic cyto- Immunostaining Assessment plasms) and the penumbra area with vacuolation of the white matter and axonal swelling, to compare affected and Immunostaining of BMPs, BMPRII, and pSMAD1/5/8 was relatively preserved areas. PML changes consisted of performed in NNC and MS, AI, and PML lesions. A demyelination with myelin-containing macrophages, bizarre neuropathologist (C.C.) blinded to the clinicopathological reactive astrocytes, and oligodendrocytes with enlarged data evaluated immunostained slides. Cells expressing the glassy nuclei reflecting viral damage. Mild neuropatholog- different studied proteins were morphologically identified as ical changes were found in NNC, mainly represented by microglia/macrophages, astrocytes, or oligodendrocytes in scarce perivascular macrophages, probably as a result of the white matter and as neurons in the NAGM proximal to mild small-vessel disease. In MS, AI, and PML lesions, the lesion. The percentage of total cells expressing BMP-2, NAGM proximal to the lesion was studied; and in NNC, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8 NAGM was studied. was assessed in the damaged white matter and, when available, in the NAGM proximal to the lesion. For each Immunohistochemistry sample, BMP-, BMPRII-, and pSMAD1/5/8-positive cells were quantified in five fields of 0.0025 mm2. To avoid bias Paraffin-embedded serial sections (4 mm thick) were due to differences in cellularity, results are expressed as the deparaffinized in xylene and rehydrated in alcohol. percentage of positive cells for each staining over total cells Endogenous peroxidase activity was blocked with in the field. hydrogen peroxide (2%; Sigma, St. Louis, MO) and methanol (70%; Merck, Darmstadt, Germany) for 20 mi- Statistical Analysis nutes. Antigen retrieval was performed in 10 mmol/L cit- rate (pH Z 6) or Tris-EDTA buffer [1 mol/L TrismaBase Because of the heterogeneous variability of the data, sta- and 1 mmol/L EDTA (Sigma), pH 9] when needed tistical analyses were performed using nonparametric tests. (Table 2). Nonspecific protein binding was blocked with The Kruskal-Wallis test was used to examine differences in 2% bovine serum albumin (Sigma) in phosphate-buffered expression between different disease conditions in the white saline. Sections were incubated overnight at 4 C with the and gray matter. Subsequently, Dunn test to adjust signifi- primary antibodies listed in Table 2. Antibody binding was cance levels was applied to evaluate the pairwise compari- visualized with appropriate secondary antibodies (Table 2) sons of interest.18 Analyses were performed with R software and 3,30 diaminobenzidine (Sigma) as the chromogenic version 3.2.3.19 The level of statistical significance was set substrate. Negative controls consisting of tissue samples at 0.05. The data are expressed as the means  SD. Figure 2 Characterization of bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-7, BMP type II receptor (BMPRII), and phosphorylated SMAD (pSMAD) 1/5/8 in the white matter of the different disease conditions studied. The expression of BMP-2, BMP-4, BMP-5, and BMP-7, BMPRII, and pSMAD1/5/8 was assessed in the damaged white matter at the edge of the lesions in chronic multiple sclerosis (MS) lesion with high inflammatory activity (CHIA), chronic MS lesion with low inflammatory activity (CLIA), and chronic inactive (CIA) MS lesions, in acute infarct (AI) lesion, in progressive multifocal leukoence- phalopathy (PML) lesions, and in normal-appearing white matter in nonneurologic control (NNC). Different cell types were morphologically identified. Arrows indicate positive immunostaining, arrowheads, microglia/macrophages; arrows with thick heads, astrocytes; arrows with thin heads, oligodendrocytes. Scale bar Z 100 mm (all images). Original magnification, 40 (all images). The American Journal of Pathology - ajp.amjpathol.org 669 Costa et al 670 ajp.amjpathol.org - The American Journal of Pathology BMPs in MS Lesions Results CHIA and CIA MS lesions. Percentage of BMP-2 (23.77%  8.10% versus 7.08%  4.42%; P < 0.05), BMPs, BMPRII, and pSMAD1/5/8 Are Expressed in BMP-4 (17.50%  7.02% versus 6.29%  5.15%; Different Cell Types in MS Lesions P < 0.05), BMP-7 (46.44%  12.65% versus 15.94%  2.90%; P < 0.05), and BMPRII First, the expression of BMP-2, BMP-4, BMP-5, and BMP- (24.92%  9.26% versus 11.37%  4.53%; P < 0.05) 7; BMPRII; and the phosphorylated form of the cytoplasmic expressing cells was higher in CHIA compared with CIA mediator SMAD1/5/8 was described through double im- MS lesions, whereas no differences in the percentage of munostaining in CHIA MS lesions. At the margins of the cells expressing BMP-5 and pSMAD1/5/8 were observed lesion, astrocytes and microglia/macrophages expressed all (Figures 4A and 5). On the other hand, the percentage of BMPs, BMPRII, and pSMAD1/5/8 (Figure 1). On the other cells positive for the studied proteins was still higher in the hand, oligodendrocytes showed only BMP-2 and BMP-7 damaged white matter of CHIA than in CLIA MS lesions: expression, and BMPRII and pSMAD1/5/8 were also pre- BMP-2 (23.77%  8.10% versus 9.26%  7.33%; sent, indicating that BMP signaling is active in this cell type P < 0.05), BMP-7 (46.44%  12.65% versus (Figure 1). In the NAGM proximal to the lesion, the neurons 29.57%  5.66%; P < 0.05), BMPRII (24.92%  9.26% expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; versus 14.78%  9.21%; P < 0.05), and pSMAD1/5/8 and pSMAD1/5/8 (Figure 1). Negative controls showed no (12.84%  4.93% versus 5.98%  4.47%; P < 0.05). The staining (Supplemental Figure S1), indicating that the differences were lost for BMP-4 when inflammatory activity immunoreactivity was specific for each primary antibody. was present in MS lesions; consistently, no differences were found in the percentage of cells stained with BMP-5 Higher Expression of BMPs, BMPRII, and pSMAD1/5/8 (Figures 4A and 5). Thus, the expression of BMPs and BMPRII is more important when the inflammatory activity in MS Lesions with High Inflammatory Activity of the chronic MS lesion is higher, whereas differences in The percentage of total BMP-2, BMP-4, BMP-5, and BMP- the expression of the cytoplasmic mediator pSMAD1/5/8 7; BMPRII; and pSMAD1/5/8 expressing cells was studied were less pronounced. in different types of MS lesions classified on the basis of In the NAGM proximal to the lesion, no differences were their inflammatory and demyelination features as CHIA, seen in the percentage of total cells expressing BMPs, CLIA, and CIA, as stated in Materials and Methods. BMPRII, or pSMAD1/5/8, except for BMP-7 in CIA Morphologically identified microglia/macrophages and compared with CHIA MS lesions (21.85%  3.64% versus astrocytes in CLIA lesions showed expression of BMP-2, 34.69%  7.05%, respectively; P < 0.05) (Figure 4B), BMP-4, and BMP-7 and BMPRII. BMP-5 was observed indicating that major changes occur in damaged white only in astrocytes, pSMAD1/5/8 in the cytoplasm of matter. microglia/macrophages, and BMP-7 in oligodendrocytes. In CIA MS lesions, BMP-2 and BMPRII were observed in Expression of BMPs, BMPRII, and pSMAD1/5/8 in astrocytes and microglia/macrophages, BMP-4, BMP-5, and Lesions of Other White MattereDamaging Diseases pSMAD1/5/8 were detected in astrocytes, and BMP-7 was expressed only in the cytoplasm of oligodendrocytes and Expression Pattern of BMP-2, BMP-4, BMP-5, BMP-7, microglia/macrophages (Figure 2). In the NAGM proximal BMPRII, and pSMAD1/5/8 in NNC, AI, and PML Lesions to the lesion, the same pattern of expression of BMPs, The expression of the studied proteins was first character- BMPRII, or pSMAD1/5/8 was observed in neurons in the ized in NNC, PML, and AI in morphologically identified different MS lesions (Figure 3). Negative controls showed CNS populations. In the normal-appearing white matter of no staining (data not shown), indicating that the immuno- NNC, the expression of BMP-2, BMP-4, BMP-5, and BMP- reactivity was specific for each primary antibody. 7, BMPRII, and pSMAD1/5/8 was detected in the oligo- It was next determined whether the inflammatory activity dendrocytes, but none of them were detected in astrocytes or in the MS lesion affected the percentage of expression of microglia/macrophages, probably because of the lack of BMPs, BMPRII, and pSMAD1/5/8. In the damaged white damage in the tissue. In the damaged white matter of PML matter, larger differences were observed when comparing lesions, BMP-2 and BMP-5 were observed only in Figure 3 Characterization of bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-7, BMP type II receptor (BMPRII), and phosphorylated SMAD (pSMAD) 1/5/8 in the gray matter of the different disease conditions studied. The expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8 was assessed in the gray matter adjacent to lesion in chronic multiple sclerosis (MS) lesion with high inflammatory activity (CHIA), chronic MS lesion with low inflammatory activity (CLIA), and chronic inactive (CIA) MS lesions, in acute infarct (AI) lesion and progressive multifocal leukoencephalopathy (PML) lesions and in the normal-appearing gray matter adjacent to the lesion in nonneurologic control (NNC). Different cell types were morphologically identified. Arrows indicate positive immunostaining, arrows with thick heads, neurons; arrows with thin heads, oligodendrocytes. Scale bar Z 100 mm (all images). Original magnification, 40 (all images). The American Journal of Pathology - ajp.amjpathol.org 671 Costa et al microglia/macrophages. BMP-4 and BMP-7 were detected oligodendrocytes. In AI lesions, BMP-2, BMP-4, BMPRII, not only in microglia/macrophages but also in astrocytes and pSMAD1/5/8 were observed in microglia/macrophages, and oligodendrocytes, BMPRII was expressed in microglia/ BMP-5 was expressed in astrocytes, and BMP-7 was macrophages and astrocytes, and pSMAD1/5/8 was observed in the cytoplasm of oligodendrocytes (Figure 2). observed mainly in microglia/macrophages and Regarding the gray matter, neurons of the NAGM prox- imal to the lesion in PML and AI and NAGM in NNC expressed all of the examined proteins, similar to the pattern observed in MS lesions (Figure 3). BMPs, BMPRII, and pSMAD1/5/8 Are Highly Expressed in Chronic Inflammatory Lesions of MS Compared with Lesions of Other White MattereDamaging Diseases It was determined whether the percentage of expression of the studied proteins was more relevant in MS lesions than in le- sions of other diseases that cause damage in the white matter. CNS areas of NNC, AI, and PML lesions were immuno- stained, and the percentage of cells that expressed BMPs, BMPRII, and pSMAD1/5/8 were compared with that in CHIA MS lesions in both the normal-appearing white matter and the NAGM (in NNC) or the damaged white matter and NAGM proximal to the lesion (in AI, PML, CHIA MS lesions). In the white matter, a higher percentage of cells that expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8 was observed in CHIA lesions than in the normal-appearing white matter of NNC (Figures 4A and 5), suggesting that the BMP signaling pathway is activated when the white matter is damaged. Moreover, BMP-2, BMPRII, and pSMAD1/5/8 were highly represented in CHIA lesions (23.77%  8.10%, 24.92%  9.26%, and 12.84%  4.93% of cells stained for BMP2, BMPRII, and pSMAD1/5/8, respectively) compared with AI (11.33%  9.33%, 7.84%  3.48%, and 3.33%  2.57%, respectively; P < 0.05) and PML (10.06%  9.12%, 5.67%  4.18%, and 0.14%  0.19%, respectively; P < 0.05) (Figure 4A). The percentage of cells that Figure 4 Differential expression of bone morphogenetic protein (BMP)- 2, BMP-4, BMP-5, BMP-7, BMP type II receptor (BMPRII), and phosphor- ylated SMAD (pSMAD) 1/5/8 in multiple sclerosis (MS) lesions with different degree of inflammatory activity and in acute infarct (AI) lesion, progressive multifocal leukoencephalopathy (PML) lesion, and non- neurologic control (NNC). The expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified in the damaged white matter and normal-appearing gray matter of MS lesions, lesions from other demyelin- ating diseases, and the normal-appearing white and gray matter of non- neurologic patients. All conditions were compared with chronic MS lesion with high inflammatory activity (CHIA), applying Kruskal-Wallis test and, subsequently, Dunn test to evaluate the pairwise comparisons of CHIA MS lesions to all of the other conditions. Quantification in the white (A) and gray (B) matter. The data are expressed as the percentage of positive cells over total nuclei. Box plots represent data from the first to the third quartile, bars represent the minimum and the maximum values, lines represent the median, and plus symbols represent the mean value. n Z 21 to 25 (A and B, CHIA lesions); n Z 6 to 9 [A and B, chronic MS lesion with low inflammatory activity (CLIA)]; n Z 4 to 8 [A and B, chronic inactive (CIA) MS lesions]; n Z 6 to 9 (A and B, PML lesions); n Z 10 to 12 (A and B, AI lesions); n Z 9 or 10 (A and B, normal-appearing white and gray matter of nonneurologic patients). *P < 0.05. 672 ajp.amjpathol.org - The American Journal of Pathology BMPs in MS Lesions Figure 5 Quantification of bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-7, BMP type II receptor (BMPRII), and phosphorylated SMAD (pSMAD) 1/5/8 expression in multiple sclerosis (MS) lesions and other demyelinating lesions. Micrographs show a general view of the expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 in the white matter of MS lesions [chronic with high inflammatory activity (CHIA) lesions, chronic with low inflammatory activity (CLIA) lesions, and chronic inactive (CIA) lesions], lesions from other demyelinating diseases [progressive multifocal leukoencephal- opathy (PML) lesions and acute infarct (AI) lesions], and normal-appearing white matter [nonneurologic control (NNC)]. Scale bar Z 200 mm (all images). Original magnification, 20 (all images). expressed BMP-5 was significantly decreased in PML The percentage of BMP-2epositive cells was higher in compared with CHIA MS lesions (1.70%  1.17% versus CHIA MS lesions than in PML (14.62%  9.17% versus 21.83%  9.81%, respectively; P < 0.05), and the per- 6.23%  3.76%, respectively; P < 0.05). In contrast, the centage of BMP-7epositive cells was lower in AI damaged percentage of BMP-4estained cells was constant in all of white matter than in CHIA MS lesions (20.77%  7.53% the pathologic conditions (Figure 4B). Similar to the results versus 46.44%  12.65%, respectively; P < 0.05) obtained in the damaged white matter, the percentage of (Figures 4A and 5). cells that expressed BMP-5 and BMP-7 in the NAGM In the NAGM proximal to the lesion, a prominent proximal to the lesion differed between AI and PML le- expression of all of the studied proteins, except for BMP-7, sions: BMP-5 was expressed in a lower percentage of cells was observed in CHIA lesions compared with NNC NAGM in PML than in CHIA (5.53%  1.58% versus (Figure 4B). Similar to the results in the damaged white 20.90%  8.83%, respectively; P < 0.05), and a lower matter, the percentage of BMPRII- and pSMAD1/5/8- percentage of BMP-7epositive cells was found in AI le- positive cells was increased in CHIA lesions sions compared with CHIA (21.90%  5.49% versus (19.23%  7.50% and 10.62%  4.90%, respectively) with 34.69%  7.50%, respectively; P < 0.05) (Figure 4B). The respect to AI (7.95%  4.33% and 4.23%  3.05%, data indicate that although BMPs are differentially respectively; P < 0.05) or PML lesions (11.00%  7.04% expressed in the gray matter when the white matter is and 0.97%  1.42%, respectively; P < 0.05) (Figure 4B). damaged, changes in their expression are more relevant The American Journal of Pathology - ajp.amjpathol.org 673 Costa et al within the lesion than in the gray matter. Overall, the infiltrates to the expression of the proteins. Thus, we hy- expression of BMPs, BMPRII, and pSMAD1/5/8 also pothesize that the immune cells that are infiltrating the CNS increased in other demyelinating conditions. In addition, the in MS lesions would be promoting a negative environment increment of BMP expression was primarily observed in the for tissue repair, although this issue has not been addressed damaged white matter and it was more pronounced in active in the present work. So, further studies should be conducted MS lesions with high inflammatory activity. to unveil the expression of BMPs in the inflammatory infiltrate and to figure out the biological relevance of im- mune cellederived BMPs in the context of MS lesions. Discussion The expression of the aforementioned proteins was compared in the damaged white matter and NAGM prox- BMPs are serious candidates to have a role in MS patho- imal to the lesion of CHIA MS lesions with that in normal- genesis because they are up-regulated in demyelinating appearing white and gray matter of NNC. All BMPs and the conditions,20,21 they tightly control neural stem cell ho- signaling pathway were found to be elevated in the diseased meostasis,9,22,23 and they hinder remyelination and repair of condition, consistent with the findings of Deininger and the damaged CNS.8,24,25 In addition, BMPs also have a role collaborators.20 It was next determined if the pattern of in the regulation of the immune system; they regulate the expression was exclusive of CHIA MS lesions by activation and differentiation of certain immune cell pop- comparing the expression of BMPs, BMPRII, and ulations (eg, regulatory T cells, CD4þ type 17 helper T pSMAD1/5/8 with that in lesions of other white cells, natural killer cells, and dendritic cells), they control matteredamaging diseases, such as AI and PML. It could the proliferation of T cells, and they have an impact in the not be concluded that the BMP increase was solely found in immune function of a variety of cell types.26,27 For instance, MS lesions; in fact, BMP expression has been previously whether they may be detrimental or beneficial in the context reported to be up-regulated in different demyelinating con- of MS is still unveiled. It probably depends on the target cell ditions and models.8,20,30 However, there was expression of (eg, which BMPRs are expressed) and on the environment pSMAD1/5/8 in MS lesions, whereas it was rarely detected in the damaged tissue (eg, the presence of inhibitory signals in the damaged white matter and NAGM proximal to the and the composition of the extracellular matrix, among lesion in AI and PML, indicating that BMP signaling is others). probably more active in MS lesions. Similarly, in myelin Herein, we studied the expression of BMPs (BMP-2, oligodendrocyte glycoproteineinduced experimental auto- BMP-4, BMP-5, and BMP-7) and the activation of the BMP immune encephalomyelitis, an animal model for MS, BMP- canonical pathway by detecting BMPRII and the phos- 4, BMP-6, and BMP-7 were up-regulated on induction of phorylated form of the cytoplasmic mediator SMAD1/5/8 in the disease.21 Remyelination failure is one of the keystones MS lesions. Oligodendrocytes expressed BMP-2 and BMP- of MS research. Although approximately 40% of active MS 7, BMPRII, and pSMAD1/5/8, indicating that BMP lesions remyelinate, translating into functional axon recov- signaling is active in these cell types. Astrocytes and ery, this process is incomplete and heterogeneous between microglia/macrophages within the lesion as well as neurons MS patients.31 Remyelination directly correlates with the of the NAGM proximal to the lesion expressed all of the recruitment of OPCs into demyelinated areas; nevertheless, BMPs, BMPRII, and pSMAD1/5/8. These results expand in approximately 70% of chronic lesions of MS, premyeli- our previous knowledge of the expression of BMPs in the nating oligodendrocytes are detected but are unable to CNS under pathologic conditions in both disease lesions remyelinate axons.15 Why remyelination fails is still un- and in animal models of MS.20,21 known, although different molecules may be involved in the It was confirmed that, when comparing MS lesions with impairment of myelin repair by inhibiting the recruitment of different degrees of inflammatory activity, the major dif- OPCs into the lesion (eg, semaphorin 3A14), inhibiting the ferences in the expression of BMPs resided in the damaged differentiation and maturation of OPCs into myelinating white matter and not in the NAGM proximal to the lesion. oligodendrocytes (eg, NogoA32), or hindering the predis- Thus, when the inflammatory activity in the lesion was position of chronically demyelinated axons to be remyeli- higher, the expression of BMP-2, BMP-7, BMPRII, and nated.15 Moreover, BMPs have been largely described as pSMAD1/5/8 was higher. By contrast, BMP-4 and BMP-5 promoters of astrogliogenesis6 and are also important in the remained elevated in all MS lesions (CHIA, CLIA, and regulation of the differentiation of oligodendrocytes, inhib- CIA). It is reported that immune cells express BMPs,28 and iting their maturation into myelinating oligodendrocytes.7 that the expression of BMPs in the peripheral blood Taking into account that OPCs in the edge of chronic le- mononuclear cells of relapsing-remitting MS patients is sions are often unable to migrate and/or maturate into deregulated and increased, especially in monocytes and T myelinating oligodendrocytes,14,33 and that demyelinated cells, with respect to healthy controls.29 The correlation of axons have probably changed their molecular composition the increased BMP signaling pathway (pSMAD1/5/8 in such a way that they are not prone to remyelinate,15 we staining) with a higher inflammatory activity in MS lesions hypothesize that the increased expression of BMPs, may be attributed in part to the contribution of inflammatory BMPRII, and pSMAD1/5/8 in MS lesions probably 674 ajp.amjpathol.org - The American Journal of Pathology BMPs in MS Lesions contributes to a poor remyelination of the damaged white differentiation of oligodendroglial-astroglial progenitor cells. J Neu- matter. Nonetheless, to further corroborate this last state- rosci 1997, 17:4112e4120 7. Grinspan JB, Edell E, Carpio DF, Beesley JS, Lavy L, Pleasure D, ment, more detailed studies would be desirable to determine Golden JA: Stage-specific effects of bone morphogenetic proteins on the oligodendroglial lineage stage of the Olig2-positive cells the oligodendrocyte lineage. J Neurobiol 2000, 43:1e17 within the lesion. In addition, an increase in the expression 8. Sabo JK, Aumann TD, Merlo D, Kilpatrick TJ, Cate HS: Remyeli- of BMP-4 and BMP-7 in a context of demyelination favored nation is altered by bone morphogenic protein signaling in demyeli- gliosis. It has also been associated with an up-regulation in nated lesions. J Neurosci 2011, 31:4504e4510 9. 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