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. 2022 Aug 1;11(15):2367.
doi: 10.3390/cells11152367.

Aberrant Expression of COX-2 and FOXG1 in Infrapatellar Fat Pad-Derived ASCs from Pre-Diabetic Donors

Benjamen T O'Donnell  1 , Tia A Monjure  1 , Sara Al-Ghadban  1 , Clara J Ives  1 , Michael P L'Ecuyer  1 , Claire Rhee  2 , Monica Romero-Lopez  2 , Zhong Li  3 , Stuart B Goodman  2 , Hang Lin  3 , Rocky S Tuan  3 , Bruce A Bunnell  1
Affiliations

Aberrant Expression of COX-2 and FOXG1 in Infrapatellar Fat Pad-Derived ASCs from Pre-Diabetic Donors

Benjamen T O'Donnell et al. Cells. .

Abstract

Osteoarthritis (OA) is a degenerative joint disease resulting in limited mobility and severe disability. Type II diabetes mellitus (T2D) is a weight-independent risk factor for OA, but a link between the two diseases has not been elucidated. Adipose stem cells (ASCs) isolated from the infrapatellar fat pad (IPFP) may be a viable regenerative cell for OA treatment. This study analyzed the expression profiles of inflammatory and adipokine-related genes in IPFP-ASCs of non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and T2D donors. Pre-T2D ASCs exhibited a substantial decrease in levels of mesenchymal markers CD90 and CD105 with no change in adipogenic differentiation compared to Non-T2D and T2D IPFP-ASCs. In addition, Cyclooxygenase-2 (COX-2), Forkhead box G1 (FOXG1) expression and prostaglandin E2 (PGE2) secretion were significantly increased in Pre-T2D IPFP-ASCs upon stimulation by interleukin-1 beta (IL-1β). Interestingly, M1 macrophages exhibited a significant reduction in expression of pro-inflammatory markers TNFα and IL-6 when co-cultured with Pre-T2D IPFP-ASCs. These data suggest that the heightened systemic inflammation associated with untreated T2D may prime the IPFP-ASCs to exhibit enhanced anti-inflammatory characteristics via suppressing the IL-6/COX-2 signaling pathway. In addition, the elevated production of PGE2 by the Pre-T2D IPFP-ASCs may also suggest the contribution of pre-diabetic conditions to the onset and progression of OA.

Keywords: COX-2; FOXG1; adipose stem cell; diabetes; infrapatellar fat pad; osteoarthritis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Decreased expression of CD90, CD73, and CD105 on Pre-T2D IPFP-ASCs. (A) Flow cytometry for CD90 and CD105 demonstrated decreased expression in Pre-T2D IPFP-ASCs compared to those from Non-T2D and T2D groups (n = 3, * p < 0.05). (B) CFU assay illustrated increased self-renewal properties in T2D IPFP-ASCs compared to Non-T2D (n = 3, * p < 0.05). (C–F) RT-qPCR for common adipokines in ASCs and adipocyte differentiated ASCs demonstrated no significant difference in adipogenic potential between Non-T2D, Pre-T2D, and T2D IPFP-ASCs (n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001). Non-T2D IPFP ASCs 7-Day is the control Group set as 1. Non-T2D: IPFP-ASCs from donors without Type II diabetes mellitus, Pre-T2D: IPFP-ASCs from donors with pre-Type II diabetes mellitus, T2D: IPFP-ASCs from donors with type II diabetes mellitus, 7D: Confluent ASCs, AQ: Adipocyte Differentiated ASCs.
Figure 2
Figure 2
No significant differences in adipogenesis between IPFP-ASCS isolated from Non-T2D, Pre-T2D, and T2D donors. (A–C) Oil Red O representative image of IPFP-ASC cultured in AdipoQual media for 28 Days (Scale Bar: 100 μm). (D) Isopropanol destaining of IPFP-ASCs cultured for 28 days in growth media or AdipoQual Media for 28 days (n = 3, * p < 0.05, ** p < 0.01,). Non-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
Figure 3
Figure 3
(A–I) Decreased expression of pro-inflammatory cytokines by IPFP-ASCs after adipocyte differentiation. (n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001). Non-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
Figure 4
Figure 4
(A–I) Increased expression of COX-2 in Pre-T2D IPFP-ASCs compared to Non-T2D and T2D IPFP-ASCs before and after IL-1β treatment. (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). Non-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
Figure 5
Figure 5
Decreased TNFα and IL-6 expression by M1 macrophages after co-culture with Pre-T2D IPFP-ASCs. (A–D) Macrophage mRNA expression determined by RT-qPCR (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Control Group: M0 Macrophages Co-cultured with Non-T2D-IPFP ASCsNon-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
Figure 6
Figure 6
Increased IL-6 and Cox-2 expression by Pre-T2D and T2D IPFP-ASCs after co-culture with M1 Macrophages. (A–H) IPFP-ASC mRNA expression determined by RT-PCR (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Reference Group: Non-T2D, 7 Day. Non-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
Figure 7
Figure 7
Increased PGE2 concentration in Pre-T2D IPFP-ASC conditioned media after induction with IL-1β. (A–D) PGE2 Concentration as determined by ELISA per total protein content as determined by BCA (n = 3, * p < 0.05). Non-T2D: Non-Type II diabetes mellitus, Pre-T2D: Pre-Type II diabetes mellitus, T2D: Type II diabetes mellitus.
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