Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells
2.2. Three-Dimensional Cell Culture Model
2.3. Gene Expression Analysis by Real-Time Polymerase Chain Reaction (RT-PCR)
2.4. Western Blotting
2.5. Enzyme-Linked Immunosorbent Assay (ELISA)
2.6. Immunofluorescence
3. Results
3.1. High Glucose (HG) Increases MCP-1, TNF-alpha, IL-1β and IL-8 Expression in Valvular Endothelial Cells (VEC) and Valvular Interstitial Cells (VIC) in the 3D Valve Model Based on Gelatin Methacrylate
3.2. High Glucose (HG) Increases Von Willebrand Factor (vWF) Expression in VEC and Cell Adhesion Molecules VCAM-1 and E-Selectin in both VEC and VIC
3.3. High Glucose Increases α4, αV and β1 Integrin Chains Gene Expression in VEC and β3 and β5 in VIC
3.4. Exposure of 3D Constructs with Valvular Cells to High Glucose Induces Changes in the Gene Expression of Extracellular Matrix Proteins and Metalloproteases
3.5. High Glucose Activates PKC Signaling Pathway in Valvular Cells
4. Discussion
- the gene expression of cytokines (MCP-1, TNF-α, IL-8 and IL-1β), of cell adhesion molecules (VCAM-1 and E-selectin) and of integrins α4, αV, β1, β3 and β5 is significantly increased by HG in VEC after 7 days of culture; moreover, the levels of MCP-1 and IL-1β secreted in the conditioned media by valvular cells from the 3D construct are significantly increased when the constructs are exposed 14 days to HG concentrations, compared to normal glucose conditions;
- the gene expression of cytokines (MCP-1, TNF-α, IL-8 and IL-1β), of cell adhesion molecules (VCAM-1 and E-selectin), of integrins β3 and β5 and of remodeling molecules (collagen III, laminin, MMP-1 and MMP-13) is significantly induced by HG in VIC mainly after 14 days of culture;
- the phosphorylated form of PKC-α is elevated in both, VEC and VIC from 3D constructs exposed to HG and is involved in the production of IL-1β by valvular cells.
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | GenBank® Accession Number | Sequences of Oligonucleotide Primers | Predicted Size (bp) |
---|---|---|---|
vWF | NM_000552 | Fw: 5′-ccttgacctcggacccttatg-3′ Rv: 5′-gatgcccgttcacaccact-3′ | 76 |
VCAM-1 | NM_080682 | Fw: 5′-gggaagatggtcgtgatcctt-3′ Rv: 5′-ttgggcatagagaccccgtt-3′ | 89 |
TNF-α | NM_000594 | Fw: 5′-ttcctcagcctcttctccttcc-3′ Rv: 5′-tgatggcagagaggaggttgac-3′ | 427 |
MCP-1 | NM_002982 | Fw: 5′-cagccagatgcaatcaatgcc-3′ Rv: 5′-tggaatcctgaacccacttct-3′ | 190 |
α-SMA | NM_001141945.2 | Fw: 5′-actgccttggtgtgtgacaa-3′ Rv: 5′-caccatcaccccctgatgtc-3′ | 120 |
IL-1β | NM_000576 | Fw: 5′-tggccctaaacagatgaagtgc-3′ Rv: 5′-tcaacacgcaggacaggtacag-3′ | 488 |
IL-8 | NM_000584 | Fw: 5′-actgagagtgattgagagtggac-3′ Rv: 5′-aaccctctgcacccagttttc-3′ | 112 |
E-selectin | NM_000450 | Fw: 5′-agagtggagcctggtcttaca-3′ Rv: 5′-cctttgctgacaataagcactgg-3′ | 77 |
α4 | NM_000885 | Fw: 5′-tacagatgcaggatcggaaaga-3′ Rv: 5′-aggttctccattagggctacc-3′ | 75 |
αV | NM_002210 | Fw: 5′-gctgtcggagatttcaatggt-3′ Rv: 5′-tctgctcgccagtaaaattgt-3′ | 136 |
β1 | NM_002211 | Fw: 5′-gtaaccaaccgtagcaaagga-3′ Rv: 5′-tcccctgatcttaatcgcaaaac-3′ | 98 |
β3 | NM_000212 | Fw: 5′-catgaaggatgatctgtggagc-3′ Rv: 5′-aatccgcaggttactggtgag-3′ | 85 |
β5 | NM_002213.5 | Fw: 5′-ggaagttcggaaacagagggt-3′ Rv: 5′-ctttcgccagccaatcttctc-3′ | 106 |
Coll III | NM_000090.3 | Fw: 5′-aggtcctgcgggtaacact-3′ Rv: 5′-actttcacccttgacaccctg-3′ | 226 |
Laminin | NM_005559.4 | Fw: 5′-gtgatggcaacagcgcaaa-3′ Rv: 5′-gacccagtgatattctctccca-3′ | 116 |
MMP-1 | NM_002421.3 | Fw:5′-aaaattacacgccagatttgcc-3′ Rv: 5′-ggtgtgacattactccagagttg-3′ | 82 |
MMP-13 | NM_002427.3 | Fw: 5′-actgagaggctccgagaaatg-3′ Rv: 5′-gaaccccgcatcttggctt-3′ | 103 |
β-actin | NM_001101.4 | Fw: 5′-catgtacgttgctatccaggc-3′ Rv: 5′-ctccttaatgtcacgcacgat-3′ | 250 |
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Ciortan, L.; Macarie, R.D.; Cecoltan, S.; Vadana, M.; Tucureanu, M.M.; Mihaila, A.C.; Droc, I.; Butoi, E.; Manduteanu, I. Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve. Polymers 2020, 12, 2786. https://doi.org/10.3390/polym12122786
Ciortan L, Macarie RD, Cecoltan S, Vadana M, Tucureanu MM, Mihaila AC, Droc I, Butoi E, Manduteanu I. Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve. Polymers. 2020; 12(12):2786. https://doi.org/10.3390/polym12122786
Chicago/Turabian StyleCiortan, Letitia, Razvan Daniel Macarie, Sergiu Cecoltan, Mihaela Vadana, Monica Madalina Tucureanu, Andreea Cristina Mihaila, Ionel Droc, Elena Butoi, and Ileana Manduteanu. 2020. "Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve" Polymers 12, no. 12: 2786. https://doi.org/10.3390/polym12122786
APA StyleCiortan, L., Macarie, R. D., Cecoltan, S., Vadana, M., Tucureanu, M. M., Mihaila, A. C., Droc, I., Butoi, E., & Manduteanu, I. (2020). Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve. Polymers, 12(12), 2786. https://doi.org/10.3390/polym12122786