BMP-7 Treatment Increases M2 Macrophage Differentiation and Reduces Inflammation and Plaque Formation in Apo E-/- Mice

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting bone morphogenetic protein-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E-/- mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-10, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.


Introduction
Atherosclerosis (ATH) was once considered a bland lipid storage disease characterized by the deposition of lipids on arterial surfaces leading to restrictive and eventual blockage of normal blood flow with consequential cardiovascular events occurring including myocardial infarction (MI) and stroke. The traditional views of ATH have crumbled with accruing and insurmountable evidence pointing to inflammation as a mediator of all stages of the disease, from its complete ligation. The incision was then sutured and the mouse remained on the heating pad until consciousness was regained. Following surgery, animals were injected intravenously with BMP-7 (200ug/kg of body weight). Injections were repeated on 2 subsequent days (total of 3 injections) and mice were left to recover for 14 days following last injection.

Preparation of Paraffin Sections and Histopathology
The left and right common carotid artery, the trachea, and the esophagus were embedded in paraffin to prepare a tissue embedded block. Serial sections (5μm) were made using a tissue processor and then placed on microscope slides (Fisher Scientific, USA). Cross sections were stained using the standard protocol for Masson's trichrome staining to visualize plaque area under a bright field light Olympus microscope. LCA images were taken and plaque area was measured using Image-J software as previously published [11].

Immunohistochemistry
To determine the presence of infiltrated monocytes and M1 and M2 macrophages in the plaque, carotid artery sections, prepared using our standard protocol, were boiled in retrieval buffer for 20 min following deparaffinization in xylene [11]. Once cooled to room temperature, the sections were washed in PBS and blocked in 10% normal goat serum (NGS). Sections were then incubated overnight at 4°C with primary antibodies against either CD14 (monocytes, Abbiotec, #25156, 1:50), iNOS (M1 macrophages, Abcam, #ab129372, 1:50), or CD206 (M2 macrophages, Abcam, #ab64693, 1:50). To detect BMP-7 receptor (BMP-7R) expression on monocytes and M2 macrophages, previously stained CD14 and CD206 sections were double stained with BMPRII (Santa Cruz, #sc-5683, 1:50). Following washings in PBS, slides were incubated with appropriate secondary antibodies for 1 hr at room temperature. Sections were mounted with Vectashield containing DAPI (4', 6-diamidino-2-phenylindole) (Vector Labs) and cover-slipped. LCA artery images (1-2 sections, n = 5 animals/group) were taken for data quantification using an Olympic IX-70 fluorescent microscope and representative images were captured using a confocal microscope. The % monocytes/artery was calculated by counting total CD14 positive cells on the artery/total arterial DAPI x 100. The % M1 macrophages/artery was calculated by counting total iNOS positive cells on the artery/total arterial DAPI x 100. The % M2 macrophages/artery was calculated by counting total CD206 positive cells on the artery/total arterial DAPI x 100. The % of BMPRII on monocytes was calculated by counting double positive CD14/BMPRII cells on the artery/total CD14 positive cells x 100. The % of BMPRII on M2 macrophages was calculated by counting double positive CD206/BMPRII cells on the artery/total CD206 positive cells x 100.

Pro-and Anti-Inflammatory Cytokine Analysis via ELISA
Pro-inflammatory cytokines (RayBiotech, MCP-1 #ELM-MCP-001 and TNF-α #ELM-TNFα-001), anti-inflammatory cytokines (RayBiotech, IL-10 #ELM-IL10-001 and IL-1RA #ELM-IL1RA-001), and BMP-7 expression (TSZ Scientific, #M7485) were quantified using commercially available ELISA kits, per manufacture's instructions, on blood serum samples previously collected from each animal. In brief, blood from each animal was collected post-sacrifice in EDTA coated tubes and centrifuged at 13000 rpm for 15 min. Supernatant containing serum was collected into separate tubes and stored at -20°C for future analysis. For each ELISA, serum was loaded into pre-coated 8-well strips for 2.5 hrs at room temperature. After washing, the detection antibody (biotinylated anti-mouse antibodies) was added followed by HRP-conjugated streptavidin and TMB One-Step substrate reagent, as provided in the kit. When the blue color developed, stop solution was added and the wells were measured at 450 nm in a microtiter plate reader (Bio Rad). Results were normalized to protein concentration and data is presented as arbitrary units (A.U.).

Systolic Velocity
A Phillips Sonos 5500 Ultrasound system was used to assess systolic blood velocity of the left common carotid artery at D14 post ligation. Briefly, mice were anesthetized under 2-4% isoflurane and 2% oxygen via nose cone. Two-dimensional (2D) images were recorded in the long axis projection using a guided B-mode with the ultrasound probe lying on the left side of the neck. Under Doppler vascular settings, three to four 2D images for systolic (SV; cm/s) blood velocity were obtained and averaged.

Statistical Analysis
Data was analyzed using One-way analysis of variance (ANOVA) followed by the Tukey Test. Data is presented as a mean ± SEM and statistical significance assigned when p<0.05.

BMP-7 Inhibits Atherosclerotic Plaque Formation in Apo E -/-Mice
Masson's trichrome stained sections were examined to determine the extent of plaque formation as a determinant of atherosclerotic lesions in all control and experimental groups (Fig 1A-1C). Quantitative analysis, at two weeks following permanent PLCA ligation, shows a significant increase in plaque formation in the PLCA group relative to sham controls (p<0.05, Fig  1D). However, when treated with BMP-7, the extent of plaque formation was significantly abrogated compared with the PLCA group (p<0.05, Fig 1D).

BMP-7 Inhibits Monocyte Infiltration Following PLCA Ligation
To extrapolate mechanisms by which BMP-7 inhibits plaque formation post-PLCA ligation, inflammatory cells, specifically monocytes, were quantified. Fig 2A contains representative photomicrographs of carotid artery sections stained with CD14, a marker for monocytes, in red (a, e, and i), DAPI, for nuclei, in blue (b, f, and j), merged images (c, g, and k), and enhanced merged images (d, h, and l). Quantitative analysis suggests a significant upregulation of infiltrated monocytes following PLCA ligation relative to the sham group (p<0.05, Fig 2A).

M1 Macrophage Differentiation Enhanced Post-PLCA Ligation in Apo E -/-Mice
To evaluate monocytic reprogramming outcomes post-infiltration into the atherosclerotic lesions, we quantified the amount of differentiated pro-inflammatory M1 macrophages. To  Fig 2B). Moreover, treatment with BMP-7 following PLCA ligation had no effect on the number of differentiated M1 macrophages relative to the PLCA group ( Fig 2B). Treatment with BMP-7 Diminishes Pro-Inflammatory Cytokine Expression Previous data suggests that macrophage subtypes have unique cytokine profiles with M1 macrophages contributing to enhanced pro-inflammatory MCP-1, TNF-α and IL-6 secretion [5].
In this regard, via ELISA analysis, pro-inflammatory cytokine expression was evaluated and data presented suggest a significant increase in circulating MCP-1 and TNF-α levels following PLCA ligation relative to the sham group (p<0.05, Fig 3A and 3B, respectively). Notably, this increase in MCP-1 and TNF-α expression was significantly abolished following treatment with BMP-7 (p<0.05, Fig 3A and 3B, respectively). Additionally, mRNA levels of TNF-α and IL-6 were evaluated by RT-PCR and our data demonstrates transcribed levels of TNF-αand IL-6 were significantly elevated in the PLCA group relative to sham control animals (p<0.05, Fig  3C and 3D, respectively). Importantly, post-PLCA ligation, administration of BMP-7 significantly blunted both TNF-α and IL-6 mRNA levels compared to the PLCA group (p<0.05, Fig  3C and 3D, respectively).

M2 Macrophage Differentiation is Enhanced in PLCA Ligated Mice Following BMP-7 Treatment
Representative images of LCA sections shown in Fig 4a-4l depict CD206 positive M2 macrophages in red (a, e, and i), total nuclei in blue (b, f, and j), merged images (c, g, and k), and enhanced merged images (d, h, and l). Although not statistically significant, a decrease in M2 macrophage was observed in the PLCA group relative to the sham group (Fig 4). Importantly, results showed significantly elevated M2 macrophage concentrations in the PLCA+BMP-7 group relative to sham and PLCA groups, suggesting BMP-7 may play a role in monocyte to M2 macrophage differentiation (p<0.05, Fig 4).
Anti-Inflammatory Cytokine Expression Increases Following BMP-7 Treatment in PLCA Apo E -/-Mice M2 macrophages secrete prototypical anti-inflammatory cytokines including IL-1RA, IL-10, and Arginase 1 [6,7]. As such, levels of IL-1RA and IL-10 were assessed via ELISA and data presented suggest a significant decline in circulating anti-inflammatory cytokines following PLCA ligation compared to sham (p<0.05. Fig 5A and 5B, respectively). Following treatment with BMP-7, a significant increase in IL-1RA and IL-10 were observed as compared to the PLCA group (p<0.05. Fig 5A and 5B, respectively). Arginase +ve cells within the LCA arteries as well as Arginase 1 mRNA expression were also evaluated. The number of Arginase +ve cells/ artery was significantly decreased following PLCA ligation compared to the sham group (p<0.05, Fig 5C). Conversely, the percentage of Arginase +ve cells/artery was significantly enhanced following treatment with BMP-7 (p<0.05, Fig 5C). Moreover, albeit transcribed Arginase 1 mRNA levels were unremarkable between PLCA and sham groups, Arginase 1 mRNA was significantly upregulated in the PLCA+BMP-7 group relative to sham and PLCA groups (p<0.05, Fig 5D).
Circulating BMP-7 is Diminished Following PLCA Ligation Next, we evaluated the levels of circulating BMP-7 to ascertain a correlation between results obtained and enhanced BMP-7 expression. The BMP-7 expression in the blood was significantly decreased in the PLCA group compared to the sham group suggesting potential interference with normal BMP-7 signaling during ATH (p<0.05, Fig 6). Importantly, following BMP-7 treatment, levels of BMP-7 were significantly upregulated in the PLCA-BMP-7 group relative to the PLCA group (p<0.05, Fig 6).  observed regarding the percentage of BMP-7R on monocytes between sham and PLCA groups, a significant increase was noted for the PLCA+BMP-7 group relative to both groups (p<0.05, Fig 7A). Additionally, the %BMP-7R on M2 macrophages was also unremarkable between sham and PLCA groups ( Fig 7B). However, in the presence of BMP-7, the number of BMP-7 receptors was significantly enhanced on M2 macrophages relative to sham and PLCA ligation operated animals (p<0.05. Fig 7B).

BMP-7 Enhances Blood Flow Post-PLCA Ligation
Finally, to understand the functional consequences of BMP-7 treatment following PLCA ligation on blood flow, systolic velocity was examined using vascular Doppler ultrasonography. Our data demonstrate a significant reduction in systolic blood flow velocity post-PLCA ligation compared with the sham group suggesting PLCA ligation interfered with normal blood flow and contributed to the development of ATH (p<0.05, Fig 8). Notably, systolic velocity was significantly improved in the PLCA+BMP-7 group compared to the PLCA group but significantly less than that of the sham group (p<0.05, Fig 8).

Discussion
Atherosclerosis is a progressive disorder characterized by damaged arterial endothelium with consequential plaque formation comprised of low-density lipoproteins (LDLs) and white blood cells (WBCs) that can lead to arterial blockage and normal blood flow interference. Experimental and clinical evidence now provided have highlighted the role of inflammation in the development and progression of atherogenesis [1,2]. In general, the non-pathogenic arterial endothelium does not support binding of WBCs. However, in early stages of ATH, areas of arterial endothelial cells initiate expression of variegated adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), which bind various types of leukocytes including monocytes and T lymphocytes. Once adhered to the endothelium, the leukocytes are then able to penetrate the intima mediated by various chemoattractants including MCP-1 [12]. Now infiltrated, the pro-inflammatory WBCs contribute to and promote a local inflammatory response. Upon infiltration, monocytes undergo reprogramming yielding two antagonistic macrophage phenotypes; "classically activated" pro-inflammatory M1 macrophages and "alternatively activated" anti-inflammatory M2 macrophages. M1 macrophages promote the inflammatory response with concomitant upregulation of various cytotoxic effectors including reactive oxygen/nitrogen intermedites and inflammatory cytokines [6,7]. Recent reports suggest an association between M1 macrophage differentiation and disease pathophysiology and progression including that of atherosclerosis, cancer, and pre-diabetes [13][14][15][16]. Contrarily, M2 macrophages, which encompass all non-classically differentiated macrophages, are implicated in salubrious mediation of the inflammatory response by bolstering remodeling, repair, and resolution, in part, through secretion of quintessential anti-inflammatory cytokines including IL-1RA, IL-10, and Arginase 1 [17,18]. As such, research is now focused on identification of various growth factors and/or small molecules, which may direct and enhance macrophage plasticity towards the M2 phenotype for the development of novel therapeutic treatments for various diseases. Recently, we reported that BMP-7 enhanced the differentiation of THP-1 monocytes into M2 macrophages with concurrent upregulation of anti-inflammatory cytokines including IL-10 and IL-1RA in vitro [9]. However, the role of BMP-7 in inflammatory mediation of atherosclerosis had yet to be investigated. In the present study, we have evaluated the effects of BMP-7 treatment on plaque formation, monocyte infiltration, M1/M2 macrophage differentiation outcomes, pro-and anti-inflammatory cytokine expression, BMP-7R expression on monocytes and M2 macrophages, and blood flow in Apo E -/mice following PLCA ligation. To the best of our Apo E -/mice, when fed a high fat diet, are regularly used as a model to examine plaque formation and subsequent ATH development, variegated cell infiltration, and vasculature adverse remodeling [19][20][21][22][23]. We, however, have recently published an Apo E -/mouse ATH model in which ATH was attained by PLCA ligation, in absence of a high fat diet, with evidence of plaque formation, lipid deposition, vascular lesions, thickening of the intima, infiltration of proinflammatory cells, and upregulation of pro-inflammatory cytokine release [11]. Using this model, we generated ATH and observed plaque formation was significantly enhanced following PLCA ligation surgery as supported by our previous findings and that of others using high fat diet models of ATH [11,20,21,24,25]. Importantly, following treatment with BMP-7, plaque formation was significantly reduced. Our data is in accordance with previously published data suggesting that BMP-7 may play a palliative, cytoprotective role in vascular proliferative  [26]. Plaque formation and associated infiltrated cell types include monocyte, vascular smooth muscle cells and fibroblasts, etc. The current study addresses the role of monocytes and macrophage differentiation with the use of BMP-7. However, it would be interesting to further examine the effects of BMP-7 on vascular smooth muscle cell proliferation and adventitial fibroblast migration into the arterial intima.
It is widely accepted that ATH is associated with a significant increase in infiltrated monocytes and M1 macrophage polarization, which was also evident in our PLCA ligated mice. However, following BMP-7 treatment, monocytic infiltration was significantly inhibited. BMP-7 administration has been reported to abrogate inflammatory cell infiltrate in other cell types including renal proximal tubular epithelial cells and alkali-injured corneal cells [27,28]. Although monocyte infiltration was inhibited following BMP-7 administration, it had no effect on M1 macrophage differentiation.
To elucidate mechanisms by which BMP-7 inhibited plaque formation following PLCA ligation surgery, M2 macrophage differentiation and anti-inflammatory cytokine release were evaluated. In the ATH model mice, M2 macrophage populations were unremarkable and antiinflammatory cytokine expression including IL-1RA, IL-10, and Arginase 1 were significantly decreased compared to sham operated mice. Importantly, following treatment with BMP-7 post-PLCA ligation, M2 macrophage differentiation was significantly enhanced compared to sham and PLCA groups and anti-inflammatory cytokine secretion, including IL-1RA, IL-10, and Arginase 1, was significantly upregulated compared to the PLCA ligated mice. Our data suggest that BMP-7 has the potential to inhibit the pro-inflammatory response that is persistent in ATH through enhanced M2 macrophage differentiation and associated anti-inflammatory mediators as previously evidenced in in vitro and in vivo models including pre-diabetic cardiomyopathy and inflammatory arthritis [9,32,33].
BMP signal transduction, in general, is propagated through ligand induced activation and oligomerization of BMP receptors (BMPRs) which in turn induces phosphorylation and activation of Smad 1/5/8 and Smad-independent cascades regulating target gene transcription [10,29,34,35]. Specifically, the BMPR2 (identified as BMP-7R within the current manuscript) is directly involved in BMP-7 ligand induced receptor activation and oligomerization and subsequent signal transduction activities. We have previously identified a correlation between BMP-7 and upregulated BMP-7R during "inflammation mimicry" assault in vitro, which resulted in decreased monocytic populations and enhanced M2 macrophage differentiation [29]. As such, BMP-7R expression was also assessed and its expression remained unchanged on monocytes and M2 macrophages for both sham and PLCA ligated animals as evidenced by co-localization of BMP-7R/CD14 and BMP-7R/CD206, respectively. However, BMP-7 application resulted in a significant increase in BMP-7R expression on both monocytes and macrophages suggesting a correlation between BMP-7R expression and enhanced BMP-7 signal transduction activity. Although we have shown in vitro that BMP-7 upregulates BMP-7R expression with consequential phosphorylation and activation of Smad 1/5/8, PI3K, Akt, and mTOR leading to enhanced M2 macrophage differentiation, future studies are warranted to extrapolate the exact signaling cascades mediated by BMP-7, which promote the salutary anti-inflammatory effects in ATH [29].
Finally, blood flow was assessed for all groups and as expected, systolic velocity was significantly disrupted following PLCA ligation. However, following BMP-7 treatment, blood flow was significantly improved in PLCA ligated Apo E -/mice. Collectively, presented data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity in a PLCA ligation model of ATH through inhibition of monocyte infiltration and associated pro-inflammatory cytokines and induction of M2 macrophage differentiation with consequential enhanced antiinflammatory cytokine expression. Furthermore, we suggest that mechanisms promoting monocyte polarization and M2 macrophage differentiation by BMP-7 involve the upregulation and activation of BMP-7R. Future studies are necessary to understand the involvement of BMP-7R-associated downstream signaling pathways.