Impact of aging and estrogen deficiency on extracellular matrix-related gene expression in rotator cuff tendons: in vitro and in vivo rat model

Abstract

Background

Injuries to the rotator cuff, including the supraspinatus, infraspinatus, subscapularis, and teres minor tendons, can cause significant pain and loss of function. Aging and postmenopause detrimentally affect the extracellular matrix (ECM) of these tendons, accelerating their degeneration. This study aimed to investigate the effects of aging and estrogen deficiency on the ECM-related gene expression of rotator cuff tendons.

Methods

Female Wistar rats aged 12, 24, and 48 weeks were used to assess ECM-related gene expression in tendon insertions from the supraspinatus and infraspinatus tendons. Estrogen deficiency was modeled in female Wistar rats by performing bilateral ovariectomy (OVX), and a sham-operated group was used as the control group. Quantitative reverse-transcription polymerase chain reaction (qPCR) and RNA-sequencing (RNA-Seq) analyses were performed on harvested rotator cuff tissues 8 weeks postoperatively. Primary cells derived from these tissues were cultured and exposed to 17β-estradiol at various concentrations (0, 1, and 10 nM) for different durations (3, 6, and 24 hours), and their responses were analyzed using qPCR.

Results

Col1a1, Col3a1, and Eln mRNA levels were significantly decreased with age. RNA-Seq analysis comparing OVX rats to sham-operated controls revealed 41 downregulated and 36 upregulated genes; Eln expression was significantly reduced, while Col1a1 and Col3a1 were unchanged; Eln expression was significantly reduced, while Col1a1 and Col3a1 were unchanged. Pathway analysis revealed enrichment in the JAK-STAT and AMPK pathways among the downregulated genes in OVX groups compared to sham-operated controls. These findings were validated by qPCR, which confirmed the RNA-Seq results and showed a significant reduction in Eln expression in the OVX group. In vitro experiments with rotator cuff–derived cells derived from 8-week-old rats exposed to 17β-estradiol showed no significant changes in Col1a1 and Col3a1 expression. However, Eln mRNA expression was significantly upregulated after 24 hours of exposure to 10 ng/mL 17β-estradiol.

Conclusion

Aging was associated with reduced expression of Col1a1, Col3a1, and Eln in rotator cuff tendons, while estrogen deficiency selectively decreased Eln expression without affecting collagen genes. In vitro, 17β-estradiol stimulation increased Eln expression in tendon-derived cells. These results suggest that aging and estrogen deficiency affect ECM gene expression through distinct mechanisms, and may contribute differently to the pathophysiology of rotator cuff degeneration associated with aging and menopause.