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Gremlin-1 potentiates the dedifferentiation of VSMC in early stages of atherosclerosis
Differentiation 109 (2019) 28–33
Contents lists available at ScienceDirect
Differentiation journal homepage: www.elsevier.com/locate/diff
Gremlin-1 potentiates the dedifferentiation of VSMC in early stages of atherosclerosis
T
Renata Silvério de Barrosa,b, Grazielle Suhett Diasa,b, Ana Paula do Rosarioa,b, Fernanda Vieira Paladinoa,b, Gabriel Herculano Lopesa, Alexandre Holthausen Camposa,b,∗ a b
Hospital Israelita Albert Einstein, São Paulo, Brazil Centro de Pesquisa Experimental, Av Albert Einstein, 627. Morumbi, 2S/Bloco A, São Paulo, SP, CEP 05651-901, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Vascular smooth muscle cell Gremlin-1 BMP4 Dedifferentiation Atherosclerosis
Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to proatherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. Results: Comparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4–6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. Conclusion: Our data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.
1. Introduction VSMC are highly specialized cells, located primarily in the tunica media of arteries. They are responsible for vessel contractility and regulation of arterial blood pressure through control of vascular tone and vessel diameter (Alexander and Owens, 2012). When terminally differentiated into a contractile phenotype, these cells present low rates of migration and proliferation and express a set of cell-specific genes. Among these genes are SM22-alpha (SM22), Smooth Muscle Myosin Heavy Chain (MHC), alpha actin (Gomez and Owens, 2012), and calponin. MHC is considered a specific gene for VSMC, but full characterization of the cells requires documentation of simultaneous expression of these additional listed genes (Babu et al., 2000). The four genes encode filament proteins typically associated
with the cytoskeleton. SM22 is a calponin-associated protein expressed from the early days of embryonic formation until the VSMC reach the mature phenotype (Li et al., 1996) and MHC has a major role in cell contractility (Owens, 1995). It is well known that in the course of atherogenesis, in response to local stimuli, VSMC lose their primary function (contractility), change to a protein-synthesizing phenotype, and take part in the formation of atherosclerotic plaques. This process of VSMC dedifferentiation (Bennett et al., 2016) which also presents morphofunctional modifications is accompanied by the reduced expression of the aforementioned specific genes (Alexander and Owens, 2012; Tabas et al., 2015). Finally, VSMC dedifferentiation is considered a key step in atherogenesis, leading to the narrowing of the lumen artery that occurs due to thickening of the tunica media and plaque growth (Tabas et al., 2015).
Abbreviations: VSMC, Vascular smooth muscle cell; BMP, bone morphogenetic protein; SM22-alpha, Smooth muscle 22- alpha; MHC, myosin heavy chain; ND, normal diet; WD, hyperlipidemic western diet; ApoE-/-, ApoE-deficient mice ∗ Corresponding author. Hospital Israelita Albert Einstein, Avenida Albert Einstein, 627. Morumbi, 2S/Bloco A, São Paulo, SP, CEP 05651-901, Brazil. E-mail address: [email protected] (A.H. Campos). https://doi.org/10.1016/j.diff.2019.08.001 Received 24 May 2019; Received in revised form 12 August 2019; Accepted 27 August 2019 Available online 30 August 2019 0301-4681/ © 2019 Published by Elsevier B.V. on behalf of International Society of Differentiation.
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control. Before treatment, cells were deprived of FBS, receiving only DMEM/F12 medium supplemented with 0.5% FBS for 24 h. RNA isolation: Total RNA from cell pellets was extracted using the Illustra RNAspin MiniRNA Isolation Kit (GE, USA) following the manufacturer's instructions. Reverse transcriptase reaction was performed using the RT-ImpromII enzyme (Promega, USA) also according to the manufacturer's instructions. Quantitative Real-Time PCR (qRT-PCR): qRT-PCR was carried out using the RotorGene thermocycler (Qiagen; Hilden, GER) and the Quantifast SYBR green MasterMix kit (Qiagen; Hilden, GER), according to manufacturer's recommendations. Expression of target genes was normalized by GAPDH mRNA levels measured concurrently. The primer list is available in supplemental Table I. Immunohistochemistry of arteries: Part of the animals were sacrificed for immunohistochemistry analysis to characterize different stages of VSMC differentiation in situ. After dissection, aortic segments were embedded in paraffin. Aorta sections were blocked with albumin and then incubated with one of the following primary antibodies: Alpha Actin (Sigma Aldrich, catalog A2547, 1:400), MHC (Abcam, catalog Ab53219, 1:100), or SM22-alpha (Abcam, catalog Ab155272, 1:100). All sections were incubated with HRP-conjugated secondary antibody and detection was performed using reagents indicated by the manufacturer. Isotypic anti-mouse IgG antibody staining was used as control. For quantification of the positive stained cells, five random regions per slide of each aorta were scanned and evaluated by a blind observer to sample identity. The CellSens software (Olympus) was used for the quantification. Statistical analysis: The numerical endpoints of the study (MHC, calponin, SM22, and Alpha Actin) were described using medians, minimum and maximum values and their frequency distributions were verified by histograms, boxplot, and quantile comparison plots. A generalized linear model, with gamma probability distribution and log binding function was fitted for each numerical endpoint. Explanatory variables “animal strain” and “diet”, as well as the interaction between lineage and diet were adjusted. Results obtained from the models were presented as estimated mean values and 95% confidence intervals, and multiple comparisons were corrected by sequential Bonferroni's method. SPSS programs were used for statistical analysis and values of p < 0.05 were considered statistically significant.
The concept of dedifferentiation is well established. However, current literature is still scarce in descriptions of the different mechanisms that influence VSMC phenotypic changes in the context of atherosclerotic lesions (Alexander and Owens, 2012). Analysis of aspects related to the phenotypic modulation that occurs in VSMC during dedifferentiation may contribute to a better understanding of the mechanisms involved in plaque formation. Bone morphogenetic proteins (BMP) are involved in cardiovascular pathology as mediators of endothelial inflammation that results from shear stress, pro-inflammatory cytokines, and oxidative stress (Yao et al., 2010). Members of the transforming growth factor-β (TGF-β) superfamily, BMP have been described as regulators of bone formation, hematopoiesis, and cell differentiation during embryogenesis (Morrell et al., 2016). In the post-embryonic phase, BMP2 is involved in vascular calcification (Liu and Jin, 2016), while BMP4 has been linked to valve pathology, pulmonary hypertension, and inflammation (Cai et al., 2012).With a role in the classic BMP pathway, Gremlin-1 inhibits the binding of BMP-2, -4, or -7 to type II receptors found on cell membranes, consequently reducing the activation of SMADS in the cytoplasm (Cai et al., 2012; Brazil et al., 2015). Our previous studies have shown that BMP-2 and -4, as well as their antagonist Gremlin-1, are expressed in VSMC from ApoE-/- mice and that their expression increased when the animals were fed a western diet (Simoes Sato et al., 2014). In addition, we also showed that BMP-2 and -4 produced by VSMC from atherosclerotic lesions induce monocyte chemotaxis (Simoes Sato et al., 2014). It has been described that Gremlin-1 is an inhibitor of Macrophage Migration Inhibitory factor (MIF) and also that it is expressed in endothelial cells from mice aorta and in human coronary arteries, which suggests a role in atherogenesis and vascular inflammation (Muller et al., 2013). Additionally, Gremlin-1 is capable of efficiently activating VEGFR2 (vascular endothelial growth factor receptor 2) and inducing angiogenesis, independently of forming complexes with BMPs (Elisabetta et al., 2016). Thus, we predict that there are several ways through which Gremlin-1 might have impact upon atherogenesis. In the present study we tested the hypothesis that changes in VSMC phenotype that occur in early stages of atherosclerosis can be influenced by BMP and/or Gremlin-1 signaling. Our findings show that both BMP4 and Gremlin-1 were effective in inducing dedifferentiation in early atherogenesis.
3. Results 2. Material and Methods 3.1. Levels of smooth muscle proteins remain unchanged in early atherogenesis
Animals and experimental design: We used animals aged 3 months. Thirty-two ApoE-deficient mice (ApoE-/-) and 41 background controls (C57BL/6) from Jackson laboratories were utilized. Animals were fed for 8 weeks a low fat control diet (ND) or a hyperlipidemic western diet (WD) (Research Diets, USA). We evaluated outcomes according to the animal strain (C57BL/6 or ApoE-/-) and the type of diet consumed (ND or WD). Morphological analysis of the aortic arches was performed through conventional histology and immunohistochemistry. All procedures performed followed guidelines of research involving animals and were approved by the local Ethics Committee. Cell culture: Primary mouse smooth muscle cells (VSMCs) from the aortic arch and thoracic aorta were obtained from explant culture and were cultivated in DMEM/F12 medium supplemented with 10% FBS. Adventitial and endothelium layers were scraped to isolate only the medial layer of the vessel. Cells were incubated at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Cells were used up to a maximum of five passages to minimize phenotypic changes due to culturing. VSMC were stimulated with recombinant BMP-2 (10 ng/mL), BMP-4 (10 ng/ mL) or Gremlin-1 (25 ng/mL) all from (Peprotech, Rocky Hill, USA) for 24 h. PDGF-BB (25 ng/mL, Peprotech, Rocky Hill, USA) a potent inducer of dedifferentiation, leading VSMC from a contractile phenotype to a synthetic proliferative profile was used as our positive control and each experiment was also performed with an “untreated” negative
We chose to start our study by characterizing in situ dedifferentiation. We analyzed histological sections from aortas obtained from the 4 groups of animals included in the study. Fig. 1 shows representative sections stained for MHC, SM22, and alpha actin proteins. Although some vascular changes occurring in the early stages of atherosclerosis were already apparent, we did not observe any difference among the four groups for the three proteins, SM22, alpha actin, and MHC, evaluated. The time period chosen for this study precedes that of histologic detection of atherosclerosis. In fact, literature reports indicate that atherosclerotic plaques in the aortic arch begin to be detected after 10 weeks on WD (Kapourchali et al., 2014). Thus, we chose to address specific gene expression after 8 weeks of diet in order to potentially detect more initial changes that may lead to the disease (see Fig. 2). 3.2. VSMC from early stages of atherogenesis respond to BMP4 but not to BMP2 In order to confirm that VSMC underwent dedifferentiation and, as our previous data indicated, that BMPs are involved in atherogenesis, we evaluated the influence of the BMP pathway on VSMC phenotype. VSMC were treated with BMP2 or BMP4 (10 ng/ml) for 24 h. An 29
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Fig. 1. SM22, MHC, and alpha actin protein expression was verified after 8 weeks of diet. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and aortic arches collected for immunohistochemistry using specific antibodies for A) SM22 B) MHC, and C) alpha actin. Graphs show quantitative analysis of the stained areas. Data are represented using means and confidence intervals. A-C) N = 3 per group.
not to the same extent as seen after the administration of PDGF-BB, BMP4 also reduced the expression of SM22 in C57BL/6, ApoE-/-, and ND, as compared to untreated (2A). BMP4 was also able to reduce the expression of alpha-actin genes in all groups evaluated (2B). There was no difference in MHC and calponin gene expression with or without the
analysis of the results was carried out by regrouping samples according either to animal strain or diet received. The positive control chosen for inducing VMSC dedifferentiation was PDGF-BB (16). Treatment with recombinant BMP2 protein did not alter the expression of any of the evaluated genes (Supplementary Fig. 1). On the other hand, although 30
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Fig. 2. Gene expression of SM22 and alpha-actin in VSMC primary cell culture after treatment with recombinant BMP4. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and their aortas collected for VSMC isolation. VSMC were treated with vehicle (untreated), BMP4 (10 ng/ml) or PDGF-BB (25 ng/ml) for 24 h and A) SM22 or B) alpha actin gene expression analyzed. Values were represented by median and confidence interval. * # versus untreated. *p < 0.05; #p < 0.001. N = 5 per group.
4. Discussion
treatment with BMP4 (Supplementary Fig. 2).
It is widely described that VSMC have a phenotypic plasticity associated with changes in levels of a group of genes that regulate protein expression. These proteins, in turn, control VSMC contractile capacity. Loss of contractility is a hallmark in atherogenesis formation. In this study we evaluated the dedifferentiation of VSMC in a murine model of atherosclerosis to monitor early stages of the processes ultimately result in the manifest disease. We observed that BMP4 influences the expression of two genes, alpha-actin and SM22, in the time points studied. In addition, Gremlin-1 acted independently of the BMP pathway, enhancing VSMC dedifferentiation as identified by decreases in all four marker genes. The degree of change was similar to that seen after the challenge with PDGF-BB, which led to changes in all evaluated genes. MHC gene had its expression increased in the C57BL/6 animals fed a western diet (data not shown). This finding was not confirmed during the analysis of MHC protein expression. It is well known that VSMC are cells with remarkable plasticity and, even in non-atherosclerotic environment, they are able to repair small damages in the arterial wal (Roostalu et al., 2018). Our experiments were performed in the initial period of the disease, in which it is still not possible to detect atherosclerotic plaques. Therefore, at that stage, it is reasonable to assume that most of VSMCs had not yet acquired a synthetic phenotype, maintaining the expression of cell differentiation genes. Post-
3.3. The BMP antagonist Gremlin-1 effectively induces dedifferentiation of VSMC derived from ApoE-/- animals To evaluate the effect of Gremlin-1, VSMC were incubated with recombinant Gremlin-1 (25 ng/ml) for 24 h. Gremlin-1 was able to reduce the expression of calponin, SM22 and alpha-actin in cells collected from ApoE-/- mice. In Fig. 3A we can observe that alpha-actin gene expression was reduced in VSMC derived from ApoE-/- animals and from animals fed WD. In Fig. 3B we documented that MHC gene expression was reduced only in the VSMC obtained from the normal diet group after Gremlin-1 treatment. The gene expression of calponin was also reduced in the VSMC of the ApoE-/- group and in the cells of the animals that received a WD (Fig. 3C). In parallel, SM22 gene expression was reduced after treatment with Gremlin-1 in cells that were obtained from ApoE-/- animals or the fed a normal diet (Fig. 3D). In Fig. 4, the effects of BMP4, Gremlin and the positive control PDGF-BB treatments on VSMC obtained from mice fed a western diet for 8 weeks in vitro are represented as a heat map. BMP2 treatment was not shown in the map, as this isoform did not modulate the expression of the genes evaluated.
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Fig. 3. Gene expression of alpha actin, MHC, calponin and SM22 in VSMC primary cell culture after treatment with Gremlin-1. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and their aortas collected for VSMC isolation. VSMC were treated with vehicle (untreated) or Gremlin (25 ng/ml) for 24 h and A) alfa actin. B) MHC. C) calponin. D) SM22 gene expression was analyzed. Values were represented by median and confidence interval. * p < 0.001. N: 5 per group.
mice, regardless of the presence of ATS or hyperlipidemia. This occurred specifically for SM22 and alpha-actin genes. We also show that the BMP antagonist Gremlin-1 was able to reduce the expression of Calponin, SM22 and alpha-actin from ApoE-/- mice regardless of the diet offered. Gremlin reduced MHC gene expression only in animals that received ND. However, the relation between Gremlin-1 and atherosclerosis remains unclear. Our research group and others have previously described Gremlin-1 expression in the aortas of ApoE-/- animals (Simoes Sato et al., 2014). We have also shown that Gremlin-1 promotes proliferation and migration of VSMC, which potentially contributes to arterial damage as a result of balloon injury (Maciel et al., 2008). Gremlin-1 has been reported as an endogenous macrophage migration inhibitory factor (MIF) antagonist. When the ratio of Gremlin-1 and MIF levels was unbalanced in the serum of patients, the risk of rupture of atherosclerotic plaques was increased (Muller and Karin, 2016). Müller et al. demonstrated that Gremlin-1 prevents the recruitment and activation of macrophages in atherosclerotic plaques by inhibiting MIF. MIF is also known as an inflammatory mediator and Gremlin-1 is able to bind with high affinity to MIF, inhibiting the release of TNF-α by macrophages, thereby attenuating the progression of the disease (MullerMuller et al., 2014).
transcriptional regulation mechanisms could also explain the discrepancy between mRNA and protein expression data. In addition, it has also been described that microRNAs can modulate the expression of differentiation genes (Tang et al., 2017; Torella et al., 2011). Although BMP2 is involved in vascular calcification (Evrard et al., 2015; Liberman et al., 2013) in activating genes related to osteoblastic differentiation (for example, RUNX2) and to VSMC dedifferentiation, in our model no effect after the administration of that protein was observed. A recent publication showed that BMP2 stimulates the transition of VSMC obtained from obese mice to an osteogenic profile. However, in that study VSMC were treated with a 5-fold higher concentration of BMP2 as compared to that employed in our experiments (Andrade, 2017). In contrast, treatment with recombinant BMP4 protein reduced SM22 and Alpha-Actin gene expression. BMP4 has also been causally associated with calcification in atherosclerotic plaques, monocyte chemoattraction, and induction of arterial hypertension (Simoes Sato et al., 2014; Koga et al., 2013; Kang et al., 2012). Our finding suggests an additional way BMP4 can take part in the formation of atherosclerotic plaques, contributing to modulation of VSMC. Furthermore, BMP4 produced effects on VSMC obtained from either C57BL/6 or ApoE-/-
Fig. 4. Heatmap of treatments performed and corresponding changes in the expression of genes evaluated. Results show genes that were significantly downregulated. Columns represent treatments performed according to animal strain and type of diet fed to them. Data represent fold change of gene expression compared to their respective untreated controls. All differences presented in the map reached statistic significance.
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References
Our study did not investigate which are the molecular mechanisms involved in dedifferentiation of VSMC after treatment with Gremlin. In addition, we did not perform other potentially valuable analyses, such as hypertrophic and migratory responses of VSMC to BMPs and/or Gremlin-1. The present study used more than 70 mice. All cell cultures were performed in triplicates, tested during a limited number of passages in order to minimize phenotypic changes in culture. In addition, the number of VSMCs isolated from the aortic arch region of the animals studied was indeed very small, making it impossible to carry out more than one experiment per group of mice. Nevertheless, we believe that one of the strengths of the present study was the use of fresh tissue, as the literature presents several studies of vascular cell differentiation that mostly employ immortalized cells. It is possible that Gremlin-1 acts on the serum response factor (SRF), myocardin, and CArG box region complex, well known regulators of VSMC classic genes (Muller et al., 2013). Studies have shown that Gremlin-1 is able to bind VEGFR2 (vascular endothelial growth factor receptor 2) and Slit proteins, inducing angiogenesis and inhibiting monocyte migration, respectively. They also demonstrated that Gremlin-1 activates signaling pathways independent on BMP proteins (Brazil et al., 2015; Chen et al., 2004). The data here presented suggest that Gremlin-1 can act in the early stages of atherosclerosis. In addition, to our knowledge, this is the first time that Gremlin-1 is described potentiating the dedifferentiation of VSMC, exclusively found in cells from ApoE-/- animals. Initially, we believed that Gremlin-1 would be able to minimize/attenuate the effects of BMP signaling, exhibiting a classic antagonist effect. However, both BMP4 and Gremlin-1 appear to contribute to dedifferentiation of VSMC in culture. Gremlin-1-induced effects were more intense in comparison to of BMP4. We believe that our results deserve further attention, particularly because the mechanisms involved in Gremlin-1 signaling were not yet identified. Taken together, our data emphasize the importance of the BMP pathway and indicate that Gremlin-1 plays a significant role in the dedifferentiation of VSMC, suggesting an action regardless of BMP signaling. Further investigations in vitro and in vivo are warranted to elucidate the mechanisms of Gremlin-1 involved in VSMC dedifferentiation.
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Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins. J. Biol. Chem. 287 (6), 3976–3986. Kapourchali, F.R., Surendiran, G., Chen, L., Uitz, E., Bahadori, B., Moghadasian, M.H., 2014. Animal models of atherosclerosis. World J. Clin. Cases 2 (5), 126–132. Koga, M., Yamauchi, A., Kanaoka, Y., Jige, R., Tsukamoto, A., Teshima, N., et al., 2013. BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages. J. Inflamm. 10 (1), 32. Li, L., Miano, J.M., Cserjesi, P., Olson, E.N., 1996. SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ. Res. 78 (2), 188–195. Liberman, M., Pesaro, A.E., Carmo, L.S., Serrano Jr., C.V., 2013. Vascular calcification: pathophysiology and clinical implications. 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Conflicts of interest The authors declare that they have no conflicts of interest to disclose. Authors' contributions AHC was responsible for the design and coordinated the present study. RSB and GSD performed the experiments. RSB and GHL were responsible for analyzing the data. RSB, AHC, APFR and FVP wrote the manuscript. RSB, AHC and APFR were responsible for discussing the results and reviewing the manuscript. Acknowledgments This study was funding by grants #2014/16461-0 from São Paulo Research Foundation (FAPESP), and S.B.I.B. Albert Einstein (SGPP #2079-14). The authors would like to thank the contribution of Dr. Anna Carla Goldberg for the specialized scientific support and thoughtful suggestions. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.diff.2019.08.001.
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Contents lists available at ScienceDirect
Differentiation journal homepage: www.elsevier.com/locate/diff
Gremlin-1 potentiates the dedifferentiation of VSMC in early stages of atherosclerosis
T
Renata Silvério de Barrosa,b, Grazielle Suhett Diasa,b, Ana Paula do Rosarioa,b, Fernanda Vieira Paladinoa,b, Gabriel Herculano Lopesa, Alexandre Holthausen Camposa,b,∗ a b
Hospital Israelita Albert Einstein, São Paulo, Brazil Centro de Pesquisa Experimental, Av Albert Einstein, 627. Morumbi, 2S/Bloco A, São Paulo, SP, CEP 05651-901, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Vascular smooth muscle cell Gremlin-1 BMP4 Dedifferentiation Atherosclerosis
Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to proatherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. Results: Comparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4–6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. Conclusion: Our data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.
1. Introduction VSMC are highly specialized cells, located primarily in the tunica media of arteries. They are responsible for vessel contractility and regulation of arterial blood pressure through control of vascular tone and vessel diameter (Alexander and Owens, 2012). When terminally differentiated into a contractile phenotype, these cells present low rates of migration and proliferation and express a set of cell-specific genes. Among these genes are SM22-alpha (SM22), Smooth Muscle Myosin Heavy Chain (MHC), alpha actin (Gomez and Owens, 2012), and calponin. MHC is considered a specific gene for VSMC, but full characterization of the cells requires documentation of simultaneous expression of these additional listed genes (Babu et al., 2000). The four genes encode filament proteins typically associated
with the cytoskeleton. SM22 is a calponin-associated protein expressed from the early days of embryonic formation until the VSMC reach the mature phenotype (Li et al., 1996) and MHC has a major role in cell contractility (Owens, 1995). It is well known that in the course of atherogenesis, in response to local stimuli, VSMC lose their primary function (contractility), change to a protein-synthesizing phenotype, and take part in the formation of atherosclerotic plaques. This process of VSMC dedifferentiation (Bennett et al., 2016) which also presents morphofunctional modifications is accompanied by the reduced expression of the aforementioned specific genes (Alexander and Owens, 2012; Tabas et al., 2015). Finally, VSMC dedifferentiation is considered a key step in atherogenesis, leading to the narrowing of the lumen artery that occurs due to thickening of the tunica media and plaque growth (Tabas et al., 2015).
Abbreviations: VSMC, Vascular smooth muscle cell; BMP, bone morphogenetic protein; SM22-alpha, Smooth muscle 22- alpha; MHC, myosin heavy chain; ND, normal diet; WD, hyperlipidemic western diet; ApoE-/-, ApoE-deficient mice ∗ Corresponding author. Hospital Israelita Albert Einstein, Avenida Albert Einstein, 627. Morumbi, 2S/Bloco A, São Paulo, SP, CEP 05651-901, Brazil. E-mail address: [email protected] (A.H. Campos). https://doi.org/10.1016/j.diff.2019.08.001 Received 24 May 2019; Received in revised form 12 August 2019; Accepted 27 August 2019 Available online 30 August 2019 0301-4681/ © 2019 Published by Elsevier B.V. on behalf of International Society of Differentiation.
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control. Before treatment, cells were deprived of FBS, receiving only DMEM/F12 medium supplemented with 0.5% FBS for 24 h. RNA isolation: Total RNA from cell pellets was extracted using the Illustra RNAspin MiniRNA Isolation Kit (GE, USA) following the manufacturer's instructions. Reverse transcriptase reaction was performed using the RT-ImpromII enzyme (Promega, USA) also according to the manufacturer's instructions. Quantitative Real-Time PCR (qRT-PCR): qRT-PCR was carried out using the RotorGene thermocycler (Qiagen; Hilden, GER) and the Quantifast SYBR green MasterMix kit (Qiagen; Hilden, GER), according to manufacturer's recommendations. Expression of target genes was normalized by GAPDH mRNA levels measured concurrently. The primer list is available in supplemental Table I. Immunohistochemistry of arteries: Part of the animals were sacrificed for immunohistochemistry analysis to characterize different stages of VSMC differentiation in situ. After dissection, aortic segments were embedded in paraffin. Aorta sections were blocked with albumin and then incubated with one of the following primary antibodies: Alpha Actin (Sigma Aldrich, catalog A2547, 1:400), MHC (Abcam, catalog Ab53219, 1:100), or SM22-alpha (Abcam, catalog Ab155272, 1:100). All sections were incubated with HRP-conjugated secondary antibody and detection was performed using reagents indicated by the manufacturer. Isotypic anti-mouse IgG antibody staining was used as control. For quantification of the positive stained cells, five random regions per slide of each aorta were scanned and evaluated by a blind observer to sample identity. The CellSens software (Olympus) was used for the quantification. Statistical analysis: The numerical endpoints of the study (MHC, calponin, SM22, and Alpha Actin) were described using medians, minimum and maximum values and their frequency distributions were verified by histograms, boxplot, and quantile comparison plots. A generalized linear model, with gamma probability distribution and log binding function was fitted for each numerical endpoint. Explanatory variables “animal strain” and “diet”, as well as the interaction between lineage and diet were adjusted. Results obtained from the models were presented as estimated mean values and 95% confidence intervals, and multiple comparisons were corrected by sequential Bonferroni's method. SPSS programs were used for statistical analysis and values of p < 0.05 were considered statistically significant.
The concept of dedifferentiation is well established. However, current literature is still scarce in descriptions of the different mechanisms that influence VSMC phenotypic changes in the context of atherosclerotic lesions (Alexander and Owens, 2012). Analysis of aspects related to the phenotypic modulation that occurs in VSMC during dedifferentiation may contribute to a better understanding of the mechanisms involved in plaque formation. Bone morphogenetic proteins (BMP) are involved in cardiovascular pathology as mediators of endothelial inflammation that results from shear stress, pro-inflammatory cytokines, and oxidative stress (Yao et al., 2010). Members of the transforming growth factor-β (TGF-β) superfamily, BMP have been described as regulators of bone formation, hematopoiesis, and cell differentiation during embryogenesis (Morrell et al., 2016). In the post-embryonic phase, BMP2 is involved in vascular calcification (Liu and Jin, 2016), while BMP4 has been linked to valve pathology, pulmonary hypertension, and inflammation (Cai et al., 2012).With a role in the classic BMP pathway, Gremlin-1 inhibits the binding of BMP-2, -4, or -7 to type II receptors found on cell membranes, consequently reducing the activation of SMADS in the cytoplasm (Cai et al., 2012; Brazil et al., 2015). Our previous studies have shown that BMP-2 and -4, as well as their antagonist Gremlin-1, are expressed in VSMC from ApoE-/- mice and that their expression increased when the animals were fed a western diet (Simoes Sato et al., 2014). In addition, we also showed that BMP-2 and -4 produced by VSMC from atherosclerotic lesions induce monocyte chemotaxis (Simoes Sato et al., 2014). It has been described that Gremlin-1 is an inhibitor of Macrophage Migration Inhibitory factor (MIF) and also that it is expressed in endothelial cells from mice aorta and in human coronary arteries, which suggests a role in atherogenesis and vascular inflammation (Muller et al., 2013). Additionally, Gremlin-1 is capable of efficiently activating VEGFR2 (vascular endothelial growth factor receptor 2) and inducing angiogenesis, independently of forming complexes with BMPs (Elisabetta et al., 2016). Thus, we predict that there are several ways through which Gremlin-1 might have impact upon atherogenesis. In the present study we tested the hypothesis that changes in VSMC phenotype that occur in early stages of atherosclerosis can be influenced by BMP and/or Gremlin-1 signaling. Our findings show that both BMP4 and Gremlin-1 were effective in inducing dedifferentiation in early atherogenesis.
3. Results 2. Material and Methods 3.1. Levels of smooth muscle proteins remain unchanged in early atherogenesis
Animals and experimental design: We used animals aged 3 months. Thirty-two ApoE-deficient mice (ApoE-/-) and 41 background controls (C57BL/6) from Jackson laboratories were utilized. Animals were fed for 8 weeks a low fat control diet (ND) or a hyperlipidemic western diet (WD) (Research Diets, USA). We evaluated outcomes according to the animal strain (C57BL/6 or ApoE-/-) and the type of diet consumed (ND or WD). Morphological analysis of the aortic arches was performed through conventional histology and immunohistochemistry. All procedures performed followed guidelines of research involving animals and were approved by the local Ethics Committee. Cell culture: Primary mouse smooth muscle cells (VSMCs) from the aortic arch and thoracic aorta were obtained from explant culture and were cultivated in DMEM/F12 medium supplemented with 10% FBS. Adventitial and endothelium layers were scraped to isolate only the medial layer of the vessel. Cells were incubated at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Cells were used up to a maximum of five passages to minimize phenotypic changes due to culturing. VSMC were stimulated with recombinant BMP-2 (10 ng/mL), BMP-4 (10 ng/ mL) or Gremlin-1 (25 ng/mL) all from (Peprotech, Rocky Hill, USA) for 24 h. PDGF-BB (25 ng/mL, Peprotech, Rocky Hill, USA) a potent inducer of dedifferentiation, leading VSMC from a contractile phenotype to a synthetic proliferative profile was used as our positive control and each experiment was also performed with an “untreated” negative
We chose to start our study by characterizing in situ dedifferentiation. We analyzed histological sections from aortas obtained from the 4 groups of animals included in the study. Fig. 1 shows representative sections stained for MHC, SM22, and alpha actin proteins. Although some vascular changes occurring in the early stages of atherosclerosis were already apparent, we did not observe any difference among the four groups for the three proteins, SM22, alpha actin, and MHC, evaluated. The time period chosen for this study precedes that of histologic detection of atherosclerosis. In fact, literature reports indicate that atherosclerotic plaques in the aortic arch begin to be detected after 10 weeks on WD (Kapourchali et al., 2014). Thus, we chose to address specific gene expression after 8 weeks of diet in order to potentially detect more initial changes that may lead to the disease (see Fig. 2). 3.2. VSMC from early stages of atherogenesis respond to BMP4 but not to BMP2 In order to confirm that VSMC underwent dedifferentiation and, as our previous data indicated, that BMPs are involved in atherogenesis, we evaluated the influence of the BMP pathway on VSMC phenotype. VSMC were treated with BMP2 or BMP4 (10 ng/ml) for 24 h. An 29
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Fig. 1. SM22, MHC, and alpha actin protein expression was verified after 8 weeks of diet. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and aortic arches collected for immunohistochemistry using specific antibodies for A) SM22 B) MHC, and C) alpha actin. Graphs show quantitative analysis of the stained areas. Data are represented using means and confidence intervals. A-C) N = 3 per group.
not to the same extent as seen after the administration of PDGF-BB, BMP4 also reduced the expression of SM22 in C57BL/6, ApoE-/-, and ND, as compared to untreated (2A). BMP4 was also able to reduce the expression of alpha-actin genes in all groups evaluated (2B). There was no difference in MHC and calponin gene expression with or without the
analysis of the results was carried out by regrouping samples according either to animal strain or diet received. The positive control chosen for inducing VMSC dedifferentiation was PDGF-BB (16). Treatment with recombinant BMP2 protein did not alter the expression of any of the evaluated genes (Supplementary Fig. 1). On the other hand, although 30
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Fig. 2. Gene expression of SM22 and alpha-actin in VSMC primary cell culture after treatment with recombinant BMP4. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and their aortas collected for VSMC isolation. VSMC were treated with vehicle (untreated), BMP4 (10 ng/ml) or PDGF-BB (25 ng/ml) for 24 h and A) SM22 or B) alpha actin gene expression analyzed. Values were represented by median and confidence interval. * # versus untreated. *p < 0.05; #p < 0.001. N = 5 per group.
4. Discussion
treatment with BMP4 (Supplementary Fig. 2).
It is widely described that VSMC have a phenotypic plasticity associated with changes in levels of a group of genes that regulate protein expression. These proteins, in turn, control VSMC contractile capacity. Loss of contractility is a hallmark in atherogenesis formation. In this study we evaluated the dedifferentiation of VSMC in a murine model of atherosclerosis to monitor early stages of the processes ultimately result in the manifest disease. We observed that BMP4 influences the expression of two genes, alpha-actin and SM22, in the time points studied. In addition, Gremlin-1 acted independently of the BMP pathway, enhancing VSMC dedifferentiation as identified by decreases in all four marker genes. The degree of change was similar to that seen after the challenge with PDGF-BB, which led to changes in all evaluated genes. MHC gene had its expression increased in the C57BL/6 animals fed a western diet (data not shown). This finding was not confirmed during the analysis of MHC protein expression. It is well known that VSMC are cells with remarkable plasticity and, even in non-atherosclerotic environment, they are able to repair small damages in the arterial wal (Roostalu et al., 2018). Our experiments were performed in the initial period of the disease, in which it is still not possible to detect atherosclerotic plaques. Therefore, at that stage, it is reasonable to assume that most of VSMCs had not yet acquired a synthetic phenotype, maintaining the expression of cell differentiation genes. Post-
3.3. The BMP antagonist Gremlin-1 effectively induces dedifferentiation of VSMC derived from ApoE-/- animals To evaluate the effect of Gremlin-1, VSMC were incubated with recombinant Gremlin-1 (25 ng/ml) for 24 h. Gremlin-1 was able to reduce the expression of calponin, SM22 and alpha-actin in cells collected from ApoE-/- mice. In Fig. 3A we can observe that alpha-actin gene expression was reduced in VSMC derived from ApoE-/- animals and from animals fed WD. In Fig. 3B we documented that MHC gene expression was reduced only in the VSMC obtained from the normal diet group after Gremlin-1 treatment. The gene expression of calponin was also reduced in the VSMC of the ApoE-/- group and in the cells of the animals that received a WD (Fig. 3C). In parallel, SM22 gene expression was reduced after treatment with Gremlin-1 in cells that were obtained from ApoE-/- animals or the fed a normal diet (Fig. 3D). In Fig. 4, the effects of BMP4, Gremlin and the positive control PDGF-BB treatments on VSMC obtained from mice fed a western diet for 8 weeks in vitro are represented as a heat map. BMP2 treatment was not shown in the map, as this isoform did not modulate the expression of the genes evaluated.
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Fig. 3. Gene expression of alpha actin, MHC, calponin and SM22 in VSMC primary cell culture after treatment with Gremlin-1. C57BL/6 and ApoE-/- mice were fed for 8 weeks a normal diet (ND) or a western diet (WD). After this period, they were sacrificed and their aortas collected for VSMC isolation. VSMC were treated with vehicle (untreated) or Gremlin (25 ng/ml) for 24 h and A) alfa actin. B) MHC. C) calponin. D) SM22 gene expression was analyzed. Values were represented by median and confidence interval. * p < 0.001. N: 5 per group.
mice, regardless of the presence of ATS or hyperlipidemia. This occurred specifically for SM22 and alpha-actin genes. We also show that the BMP antagonist Gremlin-1 was able to reduce the expression of Calponin, SM22 and alpha-actin from ApoE-/- mice regardless of the diet offered. Gremlin reduced MHC gene expression only in animals that received ND. However, the relation between Gremlin-1 and atherosclerosis remains unclear. Our research group and others have previously described Gremlin-1 expression in the aortas of ApoE-/- animals (Simoes Sato et al., 2014). We have also shown that Gremlin-1 promotes proliferation and migration of VSMC, which potentially contributes to arterial damage as a result of balloon injury (Maciel et al., 2008). Gremlin-1 has been reported as an endogenous macrophage migration inhibitory factor (MIF) antagonist. When the ratio of Gremlin-1 and MIF levels was unbalanced in the serum of patients, the risk of rupture of atherosclerotic plaques was increased (Muller and Karin, 2016). Müller et al. demonstrated that Gremlin-1 prevents the recruitment and activation of macrophages in atherosclerotic plaques by inhibiting MIF. MIF is also known as an inflammatory mediator and Gremlin-1 is able to bind with high affinity to MIF, inhibiting the release of TNF-α by macrophages, thereby attenuating the progression of the disease (MullerMuller et al., 2014).
transcriptional regulation mechanisms could also explain the discrepancy between mRNA and protein expression data. In addition, it has also been described that microRNAs can modulate the expression of differentiation genes (Tang et al., 2017; Torella et al., 2011). Although BMP2 is involved in vascular calcification (Evrard et al., 2015; Liberman et al., 2013) in activating genes related to osteoblastic differentiation (for example, RUNX2) and to VSMC dedifferentiation, in our model no effect after the administration of that protein was observed. A recent publication showed that BMP2 stimulates the transition of VSMC obtained from obese mice to an osteogenic profile. However, in that study VSMC were treated with a 5-fold higher concentration of BMP2 as compared to that employed in our experiments (Andrade, 2017). In contrast, treatment with recombinant BMP4 protein reduced SM22 and Alpha-Actin gene expression. BMP4 has also been causally associated with calcification in atherosclerotic plaques, monocyte chemoattraction, and induction of arterial hypertension (Simoes Sato et al., 2014; Koga et al., 2013; Kang et al., 2012). Our finding suggests an additional way BMP4 can take part in the formation of atherosclerotic plaques, contributing to modulation of VSMC. Furthermore, BMP4 produced effects on VSMC obtained from either C57BL/6 or ApoE-/-
Fig. 4. Heatmap of treatments performed and corresponding changes in the expression of genes evaluated. Results show genes that were significantly downregulated. Columns represent treatments performed according to animal strain and type of diet fed to them. Data represent fold change of gene expression compared to their respective untreated controls. All differences presented in the map reached statistic significance.
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References
Our study did not investigate which are the molecular mechanisms involved in dedifferentiation of VSMC after treatment with Gremlin. In addition, we did not perform other potentially valuable analyses, such as hypertrophic and migratory responses of VSMC to BMPs and/or Gremlin-1. The present study used more than 70 mice. All cell cultures were performed in triplicates, tested during a limited number of passages in order to minimize phenotypic changes in culture. In addition, the number of VSMCs isolated from the aortic arch region of the animals studied was indeed very small, making it impossible to carry out more than one experiment per group of mice. Nevertheless, we believe that one of the strengths of the present study was the use of fresh tissue, as the literature presents several studies of vascular cell differentiation that mostly employ immortalized cells. It is possible that Gremlin-1 acts on the serum response factor (SRF), myocardin, and CArG box region complex, well known regulators of VSMC classic genes (Muller et al., 2013). Studies have shown that Gremlin-1 is able to bind VEGFR2 (vascular endothelial growth factor receptor 2) and Slit proteins, inducing angiogenesis and inhibiting monocyte migration, respectively. They also demonstrated that Gremlin-1 activates signaling pathways independent on BMP proteins (Brazil et al., 2015; Chen et al., 2004). The data here presented suggest that Gremlin-1 can act in the early stages of atherosclerosis. In addition, to our knowledge, this is the first time that Gremlin-1 is described potentiating the dedifferentiation of VSMC, exclusively found in cells from ApoE-/- animals. Initially, we believed that Gremlin-1 would be able to minimize/attenuate the effects of BMP signaling, exhibiting a classic antagonist effect. However, both BMP4 and Gremlin-1 appear to contribute to dedifferentiation of VSMC in culture. Gremlin-1-induced effects were more intense in comparison to of BMP4. We believe that our results deserve further attention, particularly because the mechanisms involved in Gremlin-1 signaling were not yet identified. Taken together, our data emphasize the importance of the BMP pathway and indicate that Gremlin-1 plays a significant role in the dedifferentiation of VSMC, suggesting an action regardless of BMP signaling. Further investigations in vitro and in vivo are warranted to elucidate the mechanisms of Gremlin-1 involved in VSMC dedifferentiation.
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Conflicts of interest The authors declare that they have no conflicts of interest to disclose. Authors' contributions AHC was responsible for the design and coordinated the present study. RSB and GSD performed the experiments. RSB and GHL were responsible for analyzing the data. RSB, AHC, APFR and FVP wrote the manuscript. RSB, AHC and APFR were responsible for discussing the results and reviewing the manuscript. Acknowledgments This study was funding by grants #2014/16461-0 from São Paulo Research Foundation (FAPESP), and S.B.I.B. Albert Einstein (SGPP #2079-14). The authors would like to thank the contribution of Dr. Anna Carla Goldberg for the specialized scientific support and thoughtful suggestions. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.diff.2019.08.001.
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