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Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells
Biochemical and Biophysical Research Communications 514 (2019) 462e468
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells Aleksandra Kostina a, b, Daria Semenova a, b, c, Daria Kostina a, b, d, Vladimir Uspensky a, Anna Kostareva a, Anna Malashicheva a, b, c, * a
Almazov Federal Medical Research Centre, Saint-Petersburg, Russia Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia Saint-Petersburg State University, Saint-Petersburg, Russia d Peter the Great Saint-Petersburg Polytechnic University, Department of Medical Physics, Saint-Petersburg, Russia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2019 Accepted 27 April 2019 Available online 2 May 2019
Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in cocultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling. © 2019 Elsevier Inc. All rights reserved.
Keywords: Endothelial cells Smooth muscle cells Notch Osteogenic differentiation Calcification Cardiovascular
1. Introduction Cardiovascular calcification is a process in which mineral deposits are formed in vessel walls and heart valve leaflets and this leads to tissue stiffening, plaque rupture and cardiac complications [1,2]. No therapeutic options are available for this condition in clinic except for invasive surgeries and costly transcatheter procedures [3]. An understanding biology of calcification is needed for potential drug design. Trigger mechanisms that lead to abnormal calcification of the heart and vessels remain largely unexplored [2]. Vascular calcification can occur in the medial smooth muscle layer and in the
* Corresponding author. Almazov Federal Medical Research Centre, SaintPetersburg, Russia. E-mail addresses: [email protected], amalashicheva@gmail. com (A. Malashicheva). https://doi.org/10.1016/j.bbrc.2019.04.177 0006-291X/© 2019 Elsevier Inc. All rights reserved.
intimal layer of the vessel wall. Previously, vascular calcification has been considered to be a passive process; however, recent evidences suggest that it is an actively and tightly regulated process and it is related to bone formation by osteoblast-like cells [4]. Owing to the high plasticity smooth muscle cells have the capacity to convert from the differentiated contractile phenotype to a variety of synthetic dedifferentiated states exhibiting in some cases chondrogenesis and osteogenesis during the pathogenesis of vascular diseases [5]. The mechanisms of bone and vascular calcification seem to be similar and are connected through Notch/BMP/ TGF-b crosstalk [5,6]. The role of intercellular signaling in the cellular differentiation fate is significant. With the understanding of the general biological significance of intercellular signaling between different cellular populations, the interest of scientists has now shifted from traditional monocultures of cells to so-called co-cultures, when two different types of cells are placed together during co-culture. We have recently shown that co-culture of human aortic valve
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interstitial cells with endothelial cells leads to proosteogenic phenotype expression [7] and also we have shown that endothelial cells respond to mechanical stress by upregulation of proosteogenic factors and modulate the expression of other osteogenic factors in vascular interstitial cells. Thus, endothelial cells may contribute to vascular calcification when exposed to mechanical stress [8]. Notch is a key signaling pathway in development, ensuring cross talk between different types of cells [9]. Notch signaling in the endothelium of the vessel has been shown to mediate differentiation of underlying SMC ensuring integrity of the vessel wall [10]. Notch regulates skeletal development and homeostasis and osteoblast and osteoclast differentiation [11,12]. The aim of this study was to analyze if proosteogenic signaling is induced in the co-culture of human aortic endothelial cells (HAEC) with human aortic smooth muscle cells (SMC). We show here osteogenic gene expression induction in co-cultures of HAEC and SMC in comparison to mono-cultures and involvement of endothelial Notch signaling activation in inducing proosteogenic phenotype of co-cultures.
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of SMC and HAEC. Cells were washed with PBS and incubated with alkaline-phosphatase working solution for 15e20 min at room temperature. ALP activity appeared as blue deposition and plates were photographed with digital camera. 2.5. qPCR analysis Total RNA was extracted from cells using Extract RNA reagent (Eurogen, Russia) according to the instructions of the manufacturer. Total RNA (1 mg) was reverse transcribed with MMLV RT kit (Eurogen, Russia). Real-time PCR was performed with 1 mL cDNA and SYBRGreen PCR Mastermix (Eurogen, Russia) in the Light Cycler system using specific forward and reverse primers for target genes. Changes in target genes expression levels were calculated as fold differences using the comparative DDCT method. The mRNA levels were normalized to GAPDH mRNA. All primer sequences are available upon request. 2.6. Statistics
2. Materials and methods
qPCR data on gene expression was analyzed using Graph Pad Prism. Values are expressed as means ± SD.
2.1. Primary cultures
3. Results
To obtain smooth muscle cells cultures (SMC) the cells were isolated from the aortic wall by collagenase digestion as described previously [13]. SMC were cultured in growth medium containing DMEM (Invitrogen) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, sodium pyruvate and penicillin/streptomycin (100 mg/L) (Invitrogen). The cells were used in experiments at passages 2e7. Human aortic endothelial cells (HAEC) were isolated from the aortic wall by collagenase digestion as previously described [14]. HAEC were cultured in Endothelial Cell Medium (ECM) (ScienCell, USA). The cells were used in experiments at passages 2e5.
3.1. HAEC induce osteogenic phenotype in SMC without osteogenic medium
2.2. Co-culture of smooth muscle cells and endothelial cells SMC (100 103 cells) were plated in 6-well plates coated with 0.2% gelatin. After 24 h the 100 103 or 500 103 HAEC were added to SMC with fresh DMEM (Gibco) supplemented with 25% FBS, 2 mM L-glutamine and 100 units/ml penicillin/streptomycin. Analysis was performed 5 and 10 days after initiation of co-culture. 2.3. Magnetic cell separation CD31 also known as PECAM-1 (platelet endothelial cell adhesion molecule-1) is transmembrane glycoprotein constitutively expressed on the surface of endothelial cells. HAEC were separated from SMC using magnetic cell separation (MACS) with anti CD31þconjugated microbeads (Miltenyi Biotec, Germany) according to the manufacturer's directions. In brief, after co-culture cells were collected, resuspended in medium, mixed with FcR Blocking Reagent and CD31 MicroBeads. Then cells were loaded onto column placed in the magnetic field. The labeled CD31 þ cells (HAEC) were retained on the column while unlabeled cells (SMC) ran through. After removal of the column from the magnetic field, the magnetically retained CD31 þ cells were eluted and analyzed. 2.4. Alkaline-phosphatase staining Osteogenic process was determined by alkaline-phosphatase (ALP) activity. ALP staining was performed using Roche NBT/BCIP Tablets (Roche, Germany) 10 days after the initiation of co-culture
To reveal whether intercellular communications between endothelial and smooth muscle cells contribute to osteogenic differentiation in the absence of osteogenic medium we analyzed transcriptional level of proosteogenic markers in co-culture of SMC and HAEC. Endothelial cells were added to SMC in two different doses (Fig. 1). Expression levels of RUNX2, POSTN and COL1A1 were increased in co-cultures compared to SMC monoculture and the extent of the elevation was dependent on HAEC dosage in coculture (Fig. 1, A). BMP2, BMP4 and SOX5 mRNA were significantly elevated only in co-cultures with high amount of endothelial cells (Fig. 1, A). Alkaline phosphatase staining 10 days after initiation of co-culture confirmed that endothelial cells induced osteogenic phenotype in co-culture with SMC. Intensiveness of ALP staining correlated with the amount of HAEC in co-culture (Fig. 1, B). Osteogenic induction has not been detected in co-culture of SMC with different amount of SMC used as a control (Fig. 1, B) and confirmed that it was not increased cell density itself that increased the osteogenic capacity of SMC culture, but a specific action of endothelial cells. Thus, endothelial cells are capable to induce osteogenic phenotype in SMC without osteogenic medium. 3.2. Notch signaling is activated in co-culture of SMC with HAEC Notch pathway is a key signaling ensuring intercellular communications between adjacent cells. To explore whether Notch signaling is implicated in co-culture of HAEC and SMC we estimated mRNA level of Notch component genes after 5 days of co-culture initiation. Expression of HEY1, NOTCH1, NOTCH3 and NOTCH4 was upregulated in co-culture with high amount of HAEC compared to SMC monoculture. mRNA level of NOTCH2 was only slightly decreased in co-culture (Fig. 2). Our results suggested a possible correlation between the level of Notch activation and effectiveness of osteogenic transformation of SMC. 3.3. Notch activation promotes osteogenic transformation of SMC To elucidate the responsiveness of SMC to Notch activation we
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Fig. 1. HAEC induce osteogenic phenotype in SMC in dose-dependent manner. SMC were co-cultured with different amounts of human aortic endothelial cells (HAEC). Different amount of HAEC correspond to 100 103 и 500 103 cells per well. A. Activation of proosteogenic genes expression in co-culture of SMC with HAEC (n ¼ 5) after 5 days of initiation of co-culture. mRNA levels were analyzed by qPCR and normalized to GAPDH. B. To assess the induction of proosteogenic phenotype alkaline-phosphatase activity was verified after 10 days of initiation of co-culture.
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Fig. 2. Co-culture SMC with HAEC induces expression of Notch components genes in dose-dependent manner. Activation of Notch components genes expression in co-culture of SMC with HAEC (n ¼ 5). mRNA levels were analyzed by qPCR and normalized to GAPDH.
induced Notch signaling in SMC via transduction with different amount of lentivirus bearing Notch1-intercellular domain (NICD). We observed dose-dependent upregulation of HEY1 and ACTA2 transcription in NICD stimulated cultures (Fig. 3). We analyzed expression of proosteogenic markers and revealed dose-dependent Notch activation of RUNX2, OPN and POSTN transcription. MGP2 has been suggested to be involved in inhibition of osteoblastic events and calcification [15,16]. Accordingly, activation of Notch pathway decreased the expression of MGP2 in SMC (Fig. 3). The data confirms that Notch signaling promotes osteogenic program in SMC. 3.4. Co-culture increases expression of Notch components genes specifically in HAEC Intercellular communications in co-culture cause changes in both types of cells. To elucidate changes in gene expression induced by intercellular communications in each cell type we used anti CD31þconjugated microbeads to separate HAEC and SMC after co-culture. Gene expression analysis revealed dose-dependent downregulation of SMC specific markers ACTA2, CNN1 and SM22 in SMC after coculture with HAEC (Fig. 4, A). HEY1 and NOTCH3 were elevated in dose-dependent manner in both cell types but with significant extent exclusively in endothelial cells. mRNA level of NOTCH2 was increased in HAEC and decreased in SMC after co-culture, while NOTCH1 expression was slightly downregulated in HAEC and remained at unchanged level in SMC (Fig. 4, B). These results suggest that endothelial cells have osteoinductive effect on SMC and this effect is dependent on activation of Notch signaling in endothelial cells.
4. Discussion We show here expression of osteogenic phenotype in human aortic endothelial cells in co-culture with aortic smooth muscle cells and involvement of Notch activation derived from endothelial cells to the osteogenic induction of smooth muscle cells. Vascular calcification is an important complication of aging contributing to cardiovascular morbidity and mortality [17]. It is increasingly being accepted that vascular calcification is an active, organized, complex, and highly regulated process reflecting the plasticity of vasculature [4]. In spite of active research in this area the exact cellular and molecular contributors to vascular calcification still remain to be determined [3]. Early trigger events of pathological calcification remain unclear. The functional integrity of the endothelial layer to prevent atherogenic/procalcific processes has been known to be crucial [18], but the exact role of endothelial cells in driving SMC phenotype remains obscure. The individual functions of endothelial cells and smooth muscle cells are dependent on proper communication between these cell types. This communication begins early in embryogenesis as the blood vessels begin to form. During development, the endothelial cells differentiate from vascular progenitor cells, migrate and proliferate throughout the developing embryo; endothelial cells initiate the dialogue by sending a signal to recruit smooth muscle cells and pericytes from surrounding mesenchymal or neural crest-derived tissues. The smooth muscle cells in turn reciprocate with their own signals, and the relationship begins and continues throughout postnatal life [19].
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Fig. 3. Notch activation dose-dependent induces proosteogenic genes in SMC Smooth muscle cells (SMC) (n ¼ 10) were transduced by different amounts of lentivirus bearing Notch intercellular domain (NICD). Different amounts of virus correspond to 20 and 100 multiplicity of infection units (MOI). Expression of Notch target gene HEY1, SMC marker ACTA2, inhibitor of calcification MGP2 and proosteogenic genes in the presence of different dosages of NICD 5 days after lentiviral transduction. mRNA levels were analyzed by qPCR and normalized to GAPDH.
Our previous research has shown that endothelial cells could define proosteogenic response of mesenchymal interstitial cells in co-culture [7,8]. The ability of endothelial cells to affect mesenchymal cells has also been shown in other laboratories [20e24]. In the present work we show the influence of aortic endothelial cells on proosteogenic gene expression in aortic smooth muscle cells. This finding highlights the importance of endothelial state for aortic wall integrity. Our group has shown recently dysregulation of signaling network responsible for the stress resistance in aortic endothelial cells of the patients with aortic aneurysm [25]. At the same time SMC from the patients with aortic aneurysms have elevated proosteogeic gene expression and potential [8,26]. We hypothesize that improper signaling in endothelial cells could influence gene expression and differentiation state of underlying smooth muscle cells and this overall could lead to improper aortic wall integrity, matrix synthesis and final aortic wall rupture. In line with this suggestion is our data on phenotypic and functional changes of SMC in the patients with thoracic aortic aneurysm [13,26,27]. Notch is one of the most important signaling pathway ensuring cell-cell communication during development and postnatal life [28]. We show here implication of Notch activation in promoting proosteogenic phenotype of HAEC-SMC co-culture. The question
about the role of Notch in supporting osteogenic differentiation still remains unresolved and controversial. There is a considerable piece of data supporting its promoting role in osteogenic differentiation while at the same time some data supports the opposite view [11,29e33]. Notch is an important regulator of SMC [34]. Among all Notch receptors and ligands Notch2 and Notch3 appear to be the most important for SMC. These two receptors influence the phenotype and functions of SMC [35,36]. In our experiments elevated endothelial NOTCH2 and NOTCH3 expression was associated with gaining proosteogenic phenotype. Dysregulated Notch signaling is involved in calcification of aortic valve and in aortic aneurysm [7,37e40] and we suggest that endothelial dysregulation of Notch signaling could contribute to abnormal proosteogenic state of smooth muscle and interstitial cells in the diseased tissues. Our study highlights the importance of understanding the biology of calcification and trigger mechanisms of proosteogenic processes in cardiovascular tissues and an important role that endothelial cells play in defining the identity of smooth muscle cells. The fine-tuned relationships between the two types of cells in maintaining vessel integrity remains an important subject of future research.
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Fig. 4. Co-culture SMC with HAEC reduces expression of mesenchymal markers in SMC and activates Notch pathway in HAEC. SMC were co-cultured with different amounts of human aortic endothelial cells (HAEC). Different amount of HAEC correspond to 100 103 и 500 103 cells per well. HAEC and SMC were separated 5 days after initiation of coculture using anti CD31þ-conjugated microbeads. A. Expression level of mesenchymal markers in SMC after 5 days of co-culture with HAEC. B. Expression level of Notch components genes in SMC and HAEC after 5 days of co-culture. mRNA levels were analyzed by qPCR and normalized to GAPDH.
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Funding This work was supported by the Grant of Russian Foundation for Basic Research 18-34-00277. Conflict of interest None declared. Availability of data and materials All data generated or analyzed during this study are included in this manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.177. References [1] J.D. Hutcheson, C. Goettsch, M.A. Rogers, E. Aikawa, Revisiting Cardiovascular Calcification: A Multifaceted Disease Requiring a Multidisciplinary Approach, Semin. Cell Dev. Biol., Elsevier, 2015, pp. 68e77. [2] L.L. Demer, Y. Tintut, Inflammatory, metabolic, and genetic mechanisms of vascular CalcificationSignificance, Atertio. Thromb. Vasc. Biol. 34 (2014) 715e723. [3] M.A. Rogers, E. Aikawa, Cardiovascular calcification: artificial intelligence and big data accelerate mechanistic discovery, Nat. Rev. Cardiol. 16 (2019) 261e274. € m, Where do we stand on vascular calcification? Vasc. Pharmacol. [4] K.I. Bostro 84 (2016) 8e14. [5] C.S. Hilaire, M. Liberman, J.D. Miller, Bidirectional translation in cardiovascular calcification, Atertio. Thromb. Vasc. Biol. 36 (2016) e19ee24. [6] D.A. Towler, Commonalities between vasculature and bone, Circulation 135 (2017) 320e322. [7] A. Kostina, A. Shishkova, E. Ignatieva, O. Irtyuga, M. Bogdanova, K. Levchuk, A. Golovkin, E. Zhiduleva, V. Uspenskiy, O. Moiseeva, Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves, J. Mol. Cell. Cardiol. 114 (2018) 211e219. [8] A. Rutkovskiy, M. Lund, T.S. Siamansour, T.M. Reine, S.O. Kolset, K.L. Sand, E. Ignatieva, M.L. Gordeev, K.O. Stenslokken, G. Valen, J. Vaage, A. Malashicheva, Mechanical stress alters the expression of calcificationrelated genes in vascular interstitial and endothelial cells, Interact. Cardiovasc. Thorac. Surg. 28 (2019) 803e811. [9] E.R. Andersson, R. Sandberg, U. Lendahl, Notch signaling: simplicity in design, versatility in function, Development 138 (2011) 3593e3612. [10] A.-R. Pedrosa, A. Trindade, A.-C. Fernandes, C. Carvalho, J. Gigante, guez-Hurtado, H. Yagita, R.H. Adams, A. Duarte, Endothelial A.T. Tavares, R. Die Jagged1 antagonizes Dll4 regulation of endothelial branching and promotes vascular maturation downstream of Dll4/Notch1, Atertio. Thromb. Vasc. Biol. 35 (2015) 1134e1146. [11] E. Canalis, Notch in skeletal physiology and disease, Osteoporos. Int. 12 (2018) 2611e2621. [12] S. Zanotti, E. Canalis, Notch signaling and the skeleton, Endocr. Rev. 37 (2016) 223e253. [13] A. Malashicheva, D. Kostina, A. Kostina, O. Irtyuga, I. Voronkina, L. Smagina, E. Ignatieva, N. Gavriliuk, V. Uspensky, O. Moiseeva, Phenotypic and functional changes of endothelial and smooth muscle cells in thoracic aortic aneurysms, Int. J. Vasc. Med. (2016) 1e11, 2016. [14] A.S. Kostina, C. Uspensky Vcapital Ie, O.B. Irtyuga, E.V. Ignatieva, O. Freylikhman, N.D. Gavriliuk, O.M. Moiseeva, S. Zhuk, A. Tomilin, C.A.C. Kostareva capital A, A.B. Malashicheva, Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve, Biochim. Biophys. Acta 1862 (2016) 733e740. [15] J. O'Young, Y. Liao, Y. Xiao, J. Jalkanen, G. Lajoie, M. Karttunen, H.A. Goldberg, G.K. Hunter, Matrix Gla protein inhibits ectopic calcification by a direct interaction with hydroxyapatite crystals, J. Am. Chem. Soc. 133 (2011) 18406e18412.
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Dev. 23 (2014) 2581e2590. [22] B. Lilly, We have contact: endothelial cell-smooth muscle cell interactions, Physiology 29 (2014) 234e241. [23] J. Hjortnaes, G. Camci-Unal, J.D. Hutcheson, S.M. Jung, F.J. Schoen, J. Kluin, E. Aikawa, A. Khademhosseini, Directing valvular interstitial cell myofibroblast-like differentiation in a hybrid hydrogel platform, Adv. Healthc. Mater. 4 (2015) 121e130. [24] J. Hjortnaes, K. Shapero, C. Goettsch, J.D. Hutcheson, J. Keegan, J. Kluin, J.E. Mayer, J. Bischoff, E. Aikawa, Valvular interstitial cells suppress calcification of valvular endothelial cells, Atherosclerosis 242 (2015) 251e260. [25] A. Kostina, H. Bjork, E. Ignatieva, O. Irtyuga, V. Uspensky, D. Semenova, S. Maleki, A. Tomilin, O. Moiseeva, A. Franco-Cereceda, M. Gordeev, G. Faggian, A. Kostareva, P. Eriksson, A. Malashicheva, Notch, BMP and WNT/beta-catenin network is impaired in endothelial cells of the patients with thoracic aortic aneurysm, Atherosclerosis Suppl. 35 (2018) e6ee13. [26] E. Ignatieva, D. Kostina, O. Irtyuga, V. Uspensky, A. Golovkin, N. Gavriliuk, O. Moiseeva, A. Kostareva, A. Malashicheva, Mechanisms of smooth muscle cell differentiation are distinctly altered in thoracic aortic aneurysms associated with bicuspid or tricuspid aortic valves, Front. Physiol. 8 (2017). [27] D. Semenova, A. Kostina, O. Irtyuga, A. Shishkova, A. Malashicheva, 19Notch signaling and proosteogenic genes are activated in co-culture of human aortic valve endothelial and interstitial cells, Cardiovasc. Res. 114 (2018). S5-S5. €qvist, E.R. Andersson, Do as I say, Not (ch) as I do: lateral control of cell [28] M. Sjo fate, Dev. Biol. 447 (2019) 58e70. [29] E. Canalis, D. Bridgewater, L. Schilling, S. Zanotti, Canonical Notch activation in osteocytes causes osteopetrosis, Am. J. Physiol. Endocrinol. Metab. 310 (2016) E171eE182. [30] V. Deregowski, E. Gazzerro, L. Priest, S. Rydziel, E. 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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells Aleksandra Kostina a, b, Daria Semenova a, b, c, Daria Kostina a, b, d, Vladimir Uspensky a, Anna Kostareva a, Anna Malashicheva a, b, c, * a
Almazov Federal Medical Research Centre, Saint-Petersburg, Russia Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia Saint-Petersburg State University, Saint-Petersburg, Russia d Peter the Great Saint-Petersburg Polytechnic University, Department of Medical Physics, Saint-Petersburg, Russia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2019 Accepted 27 April 2019 Available online 2 May 2019
Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in cocultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling. © 2019 Elsevier Inc. All rights reserved.
Keywords: Endothelial cells Smooth muscle cells Notch Osteogenic differentiation Calcification Cardiovascular
1. Introduction Cardiovascular calcification is a process in which mineral deposits are formed in vessel walls and heart valve leaflets and this leads to tissue stiffening, plaque rupture and cardiac complications [1,2]. No therapeutic options are available for this condition in clinic except for invasive surgeries and costly transcatheter procedures [3]. An understanding biology of calcification is needed for potential drug design. Trigger mechanisms that lead to abnormal calcification of the heart and vessels remain largely unexplored [2]. Vascular calcification can occur in the medial smooth muscle layer and in the
* Corresponding author. Almazov Federal Medical Research Centre, SaintPetersburg, Russia. E-mail addresses: [email protected], amalashicheva@gmail. com (A. Malashicheva). https://doi.org/10.1016/j.bbrc.2019.04.177 0006-291X/© 2019 Elsevier Inc. All rights reserved.
intimal layer of the vessel wall. Previously, vascular calcification has been considered to be a passive process; however, recent evidences suggest that it is an actively and tightly regulated process and it is related to bone formation by osteoblast-like cells [4]. Owing to the high plasticity smooth muscle cells have the capacity to convert from the differentiated contractile phenotype to a variety of synthetic dedifferentiated states exhibiting in some cases chondrogenesis and osteogenesis during the pathogenesis of vascular diseases [5]. The mechanisms of bone and vascular calcification seem to be similar and are connected through Notch/BMP/ TGF-b crosstalk [5,6]. The role of intercellular signaling in the cellular differentiation fate is significant. With the understanding of the general biological significance of intercellular signaling between different cellular populations, the interest of scientists has now shifted from traditional monocultures of cells to so-called co-cultures, when two different types of cells are placed together during co-culture. We have recently shown that co-culture of human aortic valve
A. Kostina et al. / Biochemical and Biophysical Research Communications 514 (2019) 462e468
interstitial cells with endothelial cells leads to proosteogenic phenotype expression [7] and also we have shown that endothelial cells respond to mechanical stress by upregulation of proosteogenic factors and modulate the expression of other osteogenic factors in vascular interstitial cells. Thus, endothelial cells may contribute to vascular calcification when exposed to mechanical stress [8]. Notch is a key signaling pathway in development, ensuring cross talk between different types of cells [9]. Notch signaling in the endothelium of the vessel has been shown to mediate differentiation of underlying SMC ensuring integrity of the vessel wall [10]. Notch regulates skeletal development and homeostasis and osteoblast and osteoclast differentiation [11,12]. The aim of this study was to analyze if proosteogenic signaling is induced in the co-culture of human aortic endothelial cells (HAEC) with human aortic smooth muscle cells (SMC). We show here osteogenic gene expression induction in co-cultures of HAEC and SMC in comparison to mono-cultures and involvement of endothelial Notch signaling activation in inducing proosteogenic phenotype of co-cultures.
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of SMC and HAEC. Cells were washed with PBS and incubated with alkaline-phosphatase working solution for 15e20 min at room temperature. ALP activity appeared as blue deposition and plates were photographed with digital camera. 2.5. qPCR analysis Total RNA was extracted from cells using Extract RNA reagent (Eurogen, Russia) according to the instructions of the manufacturer. Total RNA (1 mg) was reverse transcribed with MMLV RT kit (Eurogen, Russia). Real-time PCR was performed with 1 mL cDNA and SYBRGreen PCR Mastermix (Eurogen, Russia) in the Light Cycler system using specific forward and reverse primers for target genes. Changes in target genes expression levels were calculated as fold differences using the comparative DDCT method. The mRNA levels were normalized to GAPDH mRNA. All primer sequences are available upon request. 2.6. Statistics
2. Materials and methods
qPCR data on gene expression was analyzed using Graph Pad Prism. Values are expressed as means ± SD.
2.1. Primary cultures
3. Results
To obtain smooth muscle cells cultures (SMC) the cells were isolated from the aortic wall by collagenase digestion as described previously [13]. SMC were cultured in growth medium containing DMEM (Invitrogen) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, sodium pyruvate and penicillin/streptomycin (100 mg/L) (Invitrogen). The cells were used in experiments at passages 2e7. Human aortic endothelial cells (HAEC) were isolated from the aortic wall by collagenase digestion as previously described [14]. HAEC were cultured in Endothelial Cell Medium (ECM) (ScienCell, USA). The cells were used in experiments at passages 2e5.
3.1. HAEC induce osteogenic phenotype in SMC without osteogenic medium
2.2. Co-culture of smooth muscle cells and endothelial cells SMC (100 103 cells) were plated in 6-well plates coated with 0.2% gelatin. After 24 h the 100 103 or 500 103 HAEC were added to SMC with fresh DMEM (Gibco) supplemented with 25% FBS, 2 mM L-glutamine and 100 units/ml penicillin/streptomycin. Analysis was performed 5 and 10 days after initiation of co-culture. 2.3. Magnetic cell separation CD31 also known as PECAM-1 (platelet endothelial cell adhesion molecule-1) is transmembrane glycoprotein constitutively expressed on the surface of endothelial cells. HAEC were separated from SMC using magnetic cell separation (MACS) with anti CD31þconjugated microbeads (Miltenyi Biotec, Germany) according to the manufacturer's directions. In brief, after co-culture cells were collected, resuspended in medium, mixed with FcR Blocking Reagent and CD31 MicroBeads. Then cells were loaded onto column placed in the magnetic field. The labeled CD31 þ cells (HAEC) were retained on the column while unlabeled cells (SMC) ran through. After removal of the column from the magnetic field, the magnetically retained CD31 þ cells were eluted and analyzed. 2.4. Alkaline-phosphatase staining Osteogenic process was determined by alkaline-phosphatase (ALP) activity. ALP staining was performed using Roche NBT/BCIP Tablets (Roche, Germany) 10 days after the initiation of co-culture
To reveal whether intercellular communications between endothelial and smooth muscle cells contribute to osteogenic differentiation in the absence of osteogenic medium we analyzed transcriptional level of proosteogenic markers in co-culture of SMC and HAEC. Endothelial cells were added to SMC in two different doses (Fig. 1). Expression levels of RUNX2, POSTN and COL1A1 were increased in co-cultures compared to SMC monoculture and the extent of the elevation was dependent on HAEC dosage in coculture (Fig. 1, A). BMP2, BMP4 and SOX5 mRNA were significantly elevated only in co-cultures with high amount of endothelial cells (Fig. 1, A). Alkaline phosphatase staining 10 days after initiation of co-culture confirmed that endothelial cells induced osteogenic phenotype in co-culture with SMC. Intensiveness of ALP staining correlated with the amount of HAEC in co-culture (Fig. 1, B). Osteogenic induction has not been detected in co-culture of SMC with different amount of SMC used as a control (Fig. 1, B) and confirmed that it was not increased cell density itself that increased the osteogenic capacity of SMC culture, but a specific action of endothelial cells. Thus, endothelial cells are capable to induce osteogenic phenotype in SMC without osteogenic medium. 3.2. Notch signaling is activated in co-culture of SMC with HAEC Notch pathway is a key signaling ensuring intercellular communications between adjacent cells. To explore whether Notch signaling is implicated in co-culture of HAEC and SMC we estimated mRNA level of Notch component genes after 5 days of co-culture initiation. Expression of HEY1, NOTCH1, NOTCH3 and NOTCH4 was upregulated in co-culture with high amount of HAEC compared to SMC monoculture. mRNA level of NOTCH2 was only slightly decreased in co-culture (Fig. 2). Our results suggested a possible correlation between the level of Notch activation and effectiveness of osteogenic transformation of SMC. 3.3. Notch activation promotes osteogenic transformation of SMC To elucidate the responsiveness of SMC to Notch activation we
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Fig. 1. HAEC induce osteogenic phenotype in SMC in dose-dependent manner. SMC were co-cultured with different amounts of human aortic endothelial cells (HAEC). Different amount of HAEC correspond to 100 103 и 500 103 cells per well. A. Activation of proosteogenic genes expression in co-culture of SMC with HAEC (n ¼ 5) after 5 days of initiation of co-culture. mRNA levels were analyzed by qPCR and normalized to GAPDH. B. To assess the induction of proosteogenic phenotype alkaline-phosphatase activity was verified after 10 days of initiation of co-culture.
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Fig. 2. Co-culture SMC with HAEC induces expression of Notch components genes in dose-dependent manner. Activation of Notch components genes expression in co-culture of SMC with HAEC (n ¼ 5). mRNA levels were analyzed by qPCR and normalized to GAPDH.
induced Notch signaling in SMC via transduction with different amount of lentivirus bearing Notch1-intercellular domain (NICD). We observed dose-dependent upregulation of HEY1 and ACTA2 transcription in NICD stimulated cultures (Fig. 3). We analyzed expression of proosteogenic markers and revealed dose-dependent Notch activation of RUNX2, OPN and POSTN transcription. MGP2 has been suggested to be involved in inhibition of osteoblastic events and calcification [15,16]. Accordingly, activation of Notch pathway decreased the expression of MGP2 in SMC (Fig. 3). The data confirms that Notch signaling promotes osteogenic program in SMC. 3.4. Co-culture increases expression of Notch components genes specifically in HAEC Intercellular communications in co-culture cause changes in both types of cells. To elucidate changes in gene expression induced by intercellular communications in each cell type we used anti CD31þconjugated microbeads to separate HAEC and SMC after co-culture. Gene expression analysis revealed dose-dependent downregulation of SMC specific markers ACTA2, CNN1 and SM22 in SMC after coculture with HAEC (Fig. 4, A). HEY1 and NOTCH3 were elevated in dose-dependent manner in both cell types but with significant extent exclusively in endothelial cells. mRNA level of NOTCH2 was increased in HAEC and decreased in SMC after co-culture, while NOTCH1 expression was slightly downregulated in HAEC and remained at unchanged level in SMC (Fig. 4, B). These results suggest that endothelial cells have osteoinductive effect on SMC and this effect is dependent on activation of Notch signaling in endothelial cells.
4. Discussion We show here expression of osteogenic phenotype in human aortic endothelial cells in co-culture with aortic smooth muscle cells and involvement of Notch activation derived from endothelial cells to the osteogenic induction of smooth muscle cells. Vascular calcification is an important complication of aging contributing to cardiovascular morbidity and mortality [17]. It is increasingly being accepted that vascular calcification is an active, organized, complex, and highly regulated process reflecting the plasticity of vasculature [4]. In spite of active research in this area the exact cellular and molecular contributors to vascular calcification still remain to be determined [3]. Early trigger events of pathological calcification remain unclear. The functional integrity of the endothelial layer to prevent atherogenic/procalcific processes has been known to be crucial [18], but the exact role of endothelial cells in driving SMC phenotype remains obscure. The individual functions of endothelial cells and smooth muscle cells are dependent on proper communication between these cell types. This communication begins early in embryogenesis as the blood vessels begin to form. During development, the endothelial cells differentiate from vascular progenitor cells, migrate and proliferate throughout the developing embryo; endothelial cells initiate the dialogue by sending a signal to recruit smooth muscle cells and pericytes from surrounding mesenchymal or neural crest-derived tissues. The smooth muscle cells in turn reciprocate with their own signals, and the relationship begins and continues throughout postnatal life [19].
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Fig. 3. Notch activation dose-dependent induces proosteogenic genes in SMC Smooth muscle cells (SMC) (n ¼ 10) were transduced by different amounts of lentivirus bearing Notch intercellular domain (NICD). Different amounts of virus correspond to 20 and 100 multiplicity of infection units (MOI). Expression of Notch target gene HEY1, SMC marker ACTA2, inhibitor of calcification MGP2 and proosteogenic genes in the presence of different dosages of NICD 5 days after lentiviral transduction. mRNA levels were analyzed by qPCR and normalized to GAPDH.
Our previous research has shown that endothelial cells could define proosteogenic response of mesenchymal interstitial cells in co-culture [7,8]. The ability of endothelial cells to affect mesenchymal cells has also been shown in other laboratories [20e24]. In the present work we show the influence of aortic endothelial cells on proosteogenic gene expression in aortic smooth muscle cells. This finding highlights the importance of endothelial state for aortic wall integrity. Our group has shown recently dysregulation of signaling network responsible for the stress resistance in aortic endothelial cells of the patients with aortic aneurysm [25]. At the same time SMC from the patients with aortic aneurysms have elevated proosteogeic gene expression and potential [8,26]. We hypothesize that improper signaling in endothelial cells could influence gene expression and differentiation state of underlying smooth muscle cells and this overall could lead to improper aortic wall integrity, matrix synthesis and final aortic wall rupture. In line with this suggestion is our data on phenotypic and functional changes of SMC in the patients with thoracic aortic aneurysm [13,26,27]. Notch is one of the most important signaling pathway ensuring cell-cell communication during development and postnatal life [28]. We show here implication of Notch activation in promoting proosteogenic phenotype of HAEC-SMC co-culture. The question
about the role of Notch in supporting osteogenic differentiation still remains unresolved and controversial. There is a considerable piece of data supporting its promoting role in osteogenic differentiation while at the same time some data supports the opposite view [11,29e33]. Notch is an important regulator of SMC [34]. Among all Notch receptors and ligands Notch2 and Notch3 appear to be the most important for SMC. These two receptors influence the phenotype and functions of SMC [35,36]. In our experiments elevated endothelial NOTCH2 and NOTCH3 expression was associated with gaining proosteogenic phenotype. Dysregulated Notch signaling is involved in calcification of aortic valve and in aortic aneurysm [7,37e40] and we suggest that endothelial dysregulation of Notch signaling could contribute to abnormal proosteogenic state of smooth muscle and interstitial cells in the diseased tissues. Our study highlights the importance of understanding the biology of calcification and trigger mechanisms of proosteogenic processes in cardiovascular tissues and an important role that endothelial cells play in defining the identity of smooth muscle cells. The fine-tuned relationships between the two types of cells in maintaining vessel integrity remains an important subject of future research.
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Fig. 4. Co-culture SMC with HAEC reduces expression of mesenchymal markers in SMC and activates Notch pathway in HAEC. SMC were co-cultured with different amounts of human aortic endothelial cells (HAEC). Different amount of HAEC correspond to 100 103 и 500 103 cells per well. HAEC and SMC were separated 5 days after initiation of coculture using anti CD31þ-conjugated microbeads. A. Expression level of mesenchymal markers in SMC after 5 days of co-culture with HAEC. B. Expression level of Notch components genes in SMC and HAEC after 5 days of co-culture. mRNA levels were analyzed by qPCR and normalized to GAPDH.
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Funding This work was supported by the Grant of Russian Foundation for Basic Research 18-34-00277. Conflict of interest None declared. Availability of data and materials All data generated or analyzed during this study are included in this manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.177. References [1] J.D. Hutcheson, C. Goettsch, M.A. Rogers, E. Aikawa, Revisiting Cardiovascular Calcification: A Multifaceted Disease Requiring a Multidisciplinary Approach, Semin. Cell Dev. Biol., Elsevier, 2015, pp. 68e77. [2] L.L. Demer, Y. Tintut, Inflammatory, metabolic, and genetic mechanisms of vascular CalcificationSignificance, Atertio. Thromb. Vasc. Biol. 34 (2014) 715e723. [3] M.A. Rogers, E. Aikawa, Cardiovascular calcification: artificial intelligence and big data accelerate mechanistic discovery, Nat. Rev. Cardiol. 16 (2019) 261e274. € m, Where do we stand on vascular calcification? Vasc. Pharmacol. [4] K.I. Bostro 84 (2016) 8e14. [5] C.S. Hilaire, M. Liberman, J.D. Miller, Bidirectional translation in cardiovascular calcification, Atertio. Thromb. Vasc. Biol. 36 (2016) e19ee24. [6] D.A. Towler, Commonalities between vasculature and bone, Circulation 135 (2017) 320e322. [7] A. Kostina, A. Shishkova, E. Ignatieva, O. Irtyuga, M. Bogdanova, K. Levchuk, A. Golovkin, E. Zhiduleva, V. Uspenskiy, O. Moiseeva, Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves, J. Mol. Cell. Cardiol. 114 (2018) 211e219. [8] A. Rutkovskiy, M. Lund, T.S. Siamansour, T.M. Reine, S.O. Kolset, K.L. Sand, E. Ignatieva, M.L. Gordeev, K.O. Stenslokken, G. Valen, J. Vaage, A. Malashicheva, Mechanical stress alters the expression of calcificationrelated genes in vascular interstitial and endothelial cells, Interact. Cardiovasc. Thorac. Surg. 28 (2019) 803e811. [9] E.R. Andersson, R. Sandberg, U. Lendahl, Notch signaling: simplicity in design, versatility in function, Development 138 (2011) 3593e3612. [10] A.-R. Pedrosa, A. Trindade, A.-C. Fernandes, C. Carvalho, J. Gigante, guez-Hurtado, H. Yagita, R.H. Adams, A. Duarte, Endothelial A.T. Tavares, R. Die Jagged1 antagonizes Dll4 regulation of endothelial branching and promotes vascular maturation downstream of Dll4/Notch1, Atertio. Thromb. Vasc. Biol. 35 (2015) 1134e1146. [11] E. Canalis, Notch in skeletal physiology and disease, Osteoporos. Int. 12 (2018) 2611e2621. [12] S. Zanotti, E. Canalis, Notch signaling and the skeleton, Endocr. Rev. 37 (2016) 223e253. [13] A. Malashicheva, D. Kostina, A. Kostina, O. Irtyuga, I. Voronkina, L. Smagina, E. Ignatieva, N. Gavriliuk, V. Uspensky, O. Moiseeva, Phenotypic and functional changes of endothelial and smooth muscle cells in thoracic aortic aneurysms, Int. J. Vasc. Med. (2016) 1e11, 2016. [14] A.S. Kostina, C. Uspensky Vcapital Ie, O.B. Irtyuga, E.V. Ignatieva, O. Freylikhman, N.D. Gavriliuk, O.M. Moiseeva, S. Zhuk, A. Tomilin, C.A.C. Kostareva capital A, A.B. Malashicheva, Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve, Biochim. Biophys. Acta 1862 (2016) 733e740. [15] J. O'Young, Y. Liao, Y. Xiao, J. Jalkanen, G. Lajoie, M. Karttunen, H.A. Goldberg, G.K. Hunter, Matrix Gla protein inhibits ectopic calcification by a direct interaction with hydroxyapatite crystals, J. Am. Chem. Soc. 133 (2011) 18406e18412.
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