The low molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synovial fibroblasts

Elizabeth D Frederick; Melissa A Hausburg; Gregory W Thomas; Leonard T Rael; Edward Brody; David Bar-Or

doi:10.1016/j.bbrep.2016.08.015

The low molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synovial fibroblasts

Biochem Biophys Rep. 2016 Aug 12:8:68-74. doi: 10.1016/j.bbrep.2016.08.015. eCollection 2016 Dec.

Authors

Elizabeth D Frederick^{1

2}, Melissa A Hausburg^{1

2}, Gregory W Thomas^{1

2}, Leonard T Rael^{1

2}, Edward Brody³, David Bar-Or^{1

2

4

5

6

7}

Affiliations

¹ Swedish Medical Center, 501 E. Hampden Ave, Englewood, CO 80113, USA.
² Ampio Pharmaceuticals Inc., 373 Inverness Pkwy, Englewood, CO 80112, USA.
³ SomaLogic Inc., 2945 Wilderness Place, Boulder, CO 80301, USA.
⁴ St. Anthony Hospital, Trauma Research Department, 11600 W. 2nd Pl, Lakewood, CO 80228, USA.
⁵ Medical Center of Plano, Trauma Research Department, 3901 W. 15th St., Plano, TX 75075, USA.
⁶ Rocky Vista University, 8401 S. Chambers Rd., Parker, CO 80134, USA.
⁷ Penrose Hospital, 2417 Cascade Ave, Colorado Springs, CO 80907, USA.

Abstract

Background: The ability to decrease inflammation and promote healing is important in the intervention and management of a variety of disease states, including osteoarthritis of the knee (OAK). Even though cyclooxygenase 2 (COX2) has an established pro-inflammatory role, evidence suggests it is also critical to the resolution that occurs after the initial activation phase of the immune response. In this study, we investigated the effects of the low molecular weight fraction of 5% human serum albumin (LMWF-5A), an agent that has proven to decrease pain and improve function in OAK patients after intra-articular injection, on the expression of COX2 and its downstream products, prostaglandins (PGs).

Methods: Fibroblast-like synoviocytes from the synovial membrane of OAK patients were treated with LMWF-5A or saline as a control with or without the addition of interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) to elicit an inflammatory response. Cells were harvested for RNA and protein at 2, 4, 8, 12, and 24 h, and media was collected at 24 h for analysis of secreted products. COX2 mRNA expression was determined by qPCR, and COX2 protein expression was determined by western blot analysis. Levels of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) in the media were quantified by competitive ELISA.

Results: In the presence of either IL-1β or TNFα, LMWF-5A increased the expression of both COX2 mRNA and protein, and this increase was significant compared to that observed with IL-1β- or TNFα-stimulated, saline-treated cells. Downstream of COX2, the levels of PGE2 were increased only in TNFα-stimulated, LMWF-5A-treated cells; however, in both IL-1β- and TNFα-stimulated cells, LMWF-5A increased the release of the anti-inflammatory prostaglandin PGD2.

Conclusion: LMWF-5A appears to trigger increased anti-inflammatory PG signaling, and this may be a primary component of its therapeutic mode of action in the treatment of OAK.

Keywords: 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; COX, cyclooxygenase; Cyclooxygenase 2; DMEM/F12, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HSA, human serum albumin; HSF-OAs, human synovial fibroblasts from patients with osteoarthritis; Human serum albumin; IL, interleukin; IgG, immunoglobulin G; Inflammation; LMWF-5A, molecular weight fraction of human serum albumin under 5000 Da; LOD, limit of detection; NF-κB, nuclear factor-κB; NSAIDs, non-steroidal anti-inflammatory drugs; OA, osteoarthritis; OAK, osteoarthritis of the knee; Osteoarthritis; PBMCs, peripheral blood mononuclear cells; PG, prostaglandin; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGH2, prostaglandin H2; PPARγ, peroxisome proliferator-activated receptor γ; PVDF, polyvinylidene fluoride; Prostaglandin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, standard error of the mean; Synovial fibroblast; TNF, tumor necrosis factor; hMSCs, human mesenchymal stem cells; qPCR, quantitative real-time polymerase chain reaction; ΔΔCT, comparative threshold cycle.