IMPLICATIONS OF PRG4/LUBRICIN IN ATTENUATING MYOFIBROBLAST-MEDIATED INTRATHORACIC ADHESION DEVELOPMENT AFTER CARDIAC SURGERY

Abstract

Dense intra-pericardial and retrosternal adhesions are an unavoidable consequence of cardiac surgery and are associated with increased mortality for patients undergoing re-sternotomies. Myofibroblasts are key mediators of fibrous adhesion synthesis and progression. We previously documented expression of proteoglycan 4 (PRG4/lubricin) in human pericardial fluid and tissue. PRG4 is a mucin-like glycoprotein that has anti-adhesive properties. Loss of PRG4 in the pericardial space following cardiac surgery may trigger adhesion development. We hypothesize PRG4, which is lost after cardiotomy, may play an anti-fibrotic role and maintain pericardial homeostasis. We examined a role for PRG4 in maintaining pericardial homeostasis using an in vitro model of wound contraction with human cardiac myofibroblasts. Human atrial cardiac myofibroblasts were isolated from atrial appendage obtained from cardiac surgery patients and embedded in a 3D collagen gel matrix. Percent contraction of the collagen matrix was used as a measure of myofibroblast activation. Activation was stimulated by pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1) (normalized contraction ratio: 1.00). In the presence of TGF-β1, addition of recombinant human lubricin (rhPRG4) at a concentration of 100 μg/mL attenuated collagen matrix contraction (0.80 ± 0.10) (unpaired t-test, N=4, p < 0.05). This effect was not a result of any toxic property of rhPRG4 as we were unable to detect any significant difference in the percentage of cells that were undergoing apoptosis between the TGF-β1 treated cells (11.56%±4.83%) and the TGF-β1+rhPRG4 treated cells (10.66%±4.16%) (unpaired t-test, N=5, p=NS). Collagen fiber alignment was used as a measure of local extracellular matrix remodeling by single human cardiac myofibroblasts embedded in 3D collagen matrices using confocal microscopy (Figure). Collagen fiber alignment by human cardiac myofibroblasts in the presence of TGF-β1 (normalized ratio: 1.17±0.03) was attenuated with the addition of rhPRG4 (0.90±0.05) (unpaired t-test, N=3, p < 0.05). Cell roundness was measured to determine effects of rhPRG4 on cell morphology. Cell roundness of human cardiac myofibroblasts in the presence of TGF-β1 (normalized ratio: 0.60±0.09) was increased with the addition of rhPRG4 (0.84±0.06) (unpaired t-test, N=3, p < 0.05). We show for the first time that rhPRG4 attenuates human cardiac myofibroblast activity and cell-mediated collagen matrix remodeling. These data support the possibility that restoring PRG4 content in the pericardial space may prevent intrathoracic adhesion development after cardiac surgery.