Induction of ICAM-1 Expression in Mouse Embryonic Fibroblasts Cultured on Fibroin-Gelatin Scaffolds

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INTRODUCTION We have previously engineered a fibroin-gelatin sponge scaffold which provides a substrate for the adhesion and proliferation of various cell types [1]. Subcutaneous injection of 200-400 μm fragments of this scaffold facilitated the regeneration of deep skin wounds in mice, apparently because of their immunomodulating activity [2]. One possible mechanism underlying the regenerative activity of gelatin-fibroin scaffolds could be an increase in the expression of adhesion molecules, which are involved in immune responses, by fibroblasts after they come into contact with the scaffold surface. Presumably, ICAM-1 is one of such molecules. Normally, only a small amount of ICAM-1 is present on fibroblasts, but changes in the microenvironment can lead to an increase in its expression. In line with this fact, the inflammatory response results in a significant increase in ICAM- 1 expression by tissue-specific fibroblasts, which in turn promotes the migration of the immune cells to the site of the inflammation [3, 4]. Moreover, ICAM-1 is important for the functioning of lymphoid organs, where this molecule facilitates contact interactions between immune, stromal, and endothelial cells [5]. Thus, reconstitution of these and other interactions mediated by the 3D environment both in vitro and in vivo is an important step in the engineering of artificial lymphoid tissue [6]. MATERIALS AND METHODS A primary culture of MEF, as well as sponge fibroin scaffolds supplemented with 30% gelatin (3D FG), was prepared as previously described [1]. For the generation of fibroin-gelatin films, the same aqueous solution as that for 3D scaffolds was used. Firboin-gelatin films or Nunc culture plastic (Thermo Fisher Scientific, USA) were used for a 2D culture. Total RNA was isolated from MEFs and analyzed according to the standard protocol using the TRI Reagent (Sigma Aldrich, USA), reverse transcription kits (Thermo Scientific, EN0521 and K1621), and a real-time PCR kit (Synthol M-440) in compliance with the manufacturer’s recommendations. The quality of the reactions was evaluated using a melting curve analysis and the electrophoresis of amplification products in 1.8% agarose gel. Photographs of the gels were prepared using the GelDoc™; XR+ System (BioRad, USA). Total RNA isolated from murine lymph nodes was used as a positive control in the analysis of Madcam1 gene expression. A quantitative PCR analysis was performed on a 7500 RT-PCR System instrument (Applied Biosystems, USA). Immunofluorescence staining was performed using αICAM1-Cy5 antibodies (KAT1), nuclear dye SYTOX orange, and FITC-phalloidin conjugate to visualize polymerized actin. The samples were embedded in Aqua-Poly/Mount (Polysciences,USA) and examined using an electron microscope Camscan Series II (Cambridge Instruments) in the SEI mode and a Nikon Eclipse Ti-E microscope with a confocal module A1 (Nikon Corp., Japan) and Apo TIRF 60×/1.49 Oil or CFI Plan Apo VC 20×/0.75 lens. RESULTS AND DISCUSSION Fibroin gelatin scaffolds have a three-dimensional porous structure characterized by complex internal and external topographies (Fig. 1A-D). Importantly, when MEFs are cultured on scaffolds, the interaction of the cell surface with the substrate occurs in various directions (Fig. 1D, 1E). Cell distribution on the surface of a 3D scaffold is also shown in Fig. 2A. Since adhesion molecules play a key role in cell-to-cell and cell-to-extracellular matrix interactions, the expression of the ICAM-1 adhesion molecule was analyzed in 3D and 2D cultures to study the effect of culture conditions on the MEF phenotype. It is known that the cytoplasmic domain of the ICAM-1 molecule interacts with the actin cytoskeleton [7], and ICAM-1 molecules clustering induces their association with actin-binding adapter proteins and binding to the F-actin cytoskeleton [8]. We hypothesized that the cytoskeleton reorganization caused by MEF cultured on 3D fibroin gelatin scaffolds can alter the ICAM-1 expression. Indeed, long-term MEF culture on fibroin gelatin scaffolds, but not on culture plastic, resulted is a significant increase in the Icam1 gene expression (Fig. 2B). In full agreement with the gene expression data, bright staining of MEF with αICAM1 antibodies was observed only under 3D culture conditions (Fig. 2B), while in 2D cultures on FG films or on a glass surface, almost no staining was observed (Fig. 2D). The presence of a very weak signal was due to baseline Icam1 mRNA expression in 2D cultures (Fig. 2B). Next, we analyzed the gene expression of another adhesion molecule, MAdCAM-1, in order to verify the specificity of the observed effect for ICAM-1 expression. Similarly to ICAM-1, MAdCAM-1 is expressed on stromal and endothelial cells and it is one of the key participants in the immune cell migration to lymphoid organs and barrier tissues, but it is characterized by a specific induction associated with cytokine signaling [9]. We did not detect Madcam1 gene expression in MEFs cultured on scaffolds, which indicates the selectivity of the effect of 3D-culture conditions on the expression of the genes that encode adhesion molecules (Fig. 2E). It is known that the promoter region of the Icam-1 gene contains three binding sites for the AP-1 transcription factor, which is involved in its regulation [10]. Thus, one possible mechanism of ICAM-1 induction in MEF might be the altered activity of AP-1. The analysis of the expression of the genes that encode AP-1 subunits (Fos, Jun, Jund, Junb) (Fig. 3) demonstrated a significant increase in the level of Fos and Junb expression under 3D culture conditions as compared to 2D. At the same time, Jun and Jund expression did not depend on the culture conditions and did not change significantly. Further investigations should explore which signaling pathways, starting from the mechanistic reception of 3D scaffolds by fibroblasts or intercellular interactions, could lead to AP-1 induction, followed by ICAM-1 overexpression. In addition, other transcription factors could potentially be involved in the induction of ICAM-1 overexpression in MEF. For example, it is known that NF-χB can regulate ICAM-1 expression [11]. CONCLUSION The culture of MEF in 3D fibroin-gelatin scaffolds leads to a significant increase in the ICAM-1 expression. The increase in ICAM-1 expression is associated with the 3D structure of the scaffold rather than the influence of fibroin degradation products, since culturing on 2D fibroin films did not affect the expression of ICAM-1 in MEF. Increased ICAM-1 expression is associated with increased expression of AP-1 genes, Fos and Junb, but not Jun and Jund.

About the authors

M. A. Nosenko

Lomonosov Moscow State University; Engelhardt Insitute of Molecular Biology; German Rheumatism Research Center

Email: mmoisenovich@mail.ru
Russian Federation

N. V. Maluchenko

Lomonosov Moscow State University

Email: mmoisenovich@mail.ru
Russian Federation

M. S. Drutskaya

Lomonosov Moscow State University; Engelhardt Insitute of Molecular Biology

Email: mmoisenovich@mail.ru
Russian Federation

A. Y. Arkhipova

Lomonosov Moscow State University

Email: mmoisenovich@mail.ru
Russian Federation

I. I. Agapov

Laboratory of Bionanotechnologies, Shumakov Federal Scientific Center for Transplantology and Artificial Organs

Email: mmoisenovich@mail.ru
Russian Federation

S. A. Nedospasov

Lomonosov Moscow State University; Engelhardt Insitute of Molecular Biology; German Rheumatism Research Center

Email: mmoisenovich@mail.ru
Russian Federation

M. M. Moisenovich

Lomonosov Moscow State University

Author for correspondence.
Email: mmoisenovich@mail.ru
Russian Federation

References

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