Cyclic mechanical stretching of human patellar tendon fibroblasts: activation of JNK and modulation of apoptosis

Abstract

Accelerated rehabilitation after tendon, ligament and bone injuries is widely accepted to avoid adverse effects of immobilization. However, progressive rehabilitation may also lead to an excessive inflammatory soft tissue response and often leads to structural and functional problems such as excessive scarring. The equivalent at the molecular/cellular level is in part the regulation of the sensitive homeostasis between proliferation and apoptosis. However, little attention has been paid to this aspect of tendon pathogenesis. This study investigated the response profile of human tendon fibroblasts in terms of apoptosis and anticipated alteration of Jun N-terminal kinase (JNK) activation to cyclic mechanical stretching. Human tendon fibroblasts of six patients were stretched for 15 or 60 min with 1 Hz and an elongation of 5%. Activation of stress-activated protein kinase (SAPK)/JNK was measured by western blot analysis. Apoptotic cells were determined in the stretched cells and in controls by annexin-V staining and detection by flow cytometry. Additionally DNA laddering was determined by ligation-mediated (LM) polymerase chain reaction (PCR). Application of 15 and 60 min stretch increased activation of SAPK/JNK at a maximum after 60 min. However, JNK activation after the longer stretch period 50% less than after the shorter stretch period (15 min). The apoptosis rate was correspondingly increased after short stretch application but not after longer stretch. This might be caused by an inactivation of the activated JNKs by cell protection mechanisms. The findings suggest that mechanical stretching directly activates intracellular signaling pathways, which in turn induce apoptosis. The longer stretch period resulted in a decreased apoptotic rate due to development of stress tolerance. This might be caused by heat-shock protein mediated suppression of JNK activation. This novel observation is an important issue, as defined mechanical stretching, depending on its duration, modulates apoptosis and thus affects tendon remodeling.

Flavones Inhibit LPS-Induced Atrogin-1/MAFbx Expression in Mouse C2C12 Skeletal Myotubes

“…The activation of the JNK signaling cascade generally results in apoptosis, although this pathway has also been shown to promote cell survival under specific conditions (25). In addition, the cyclic mechanical stretching of human patellar tendon fibroblasts activates JNK and modulates apoptosis (26). Although the effects of flavonoids on the NF-kB inflammatory pathway have received considerable attention, the expression of the inflammatory cytokine IL-6 is mediated by an activator protein-1 (AP-1) regulatory element in addition to NF-kB (27).…”

The Basic Science of Tendinopathy

“…These two pathways are associated with oxidative stress. Oxidative stress can be induced during high dose cyclic strain in human and animal tendon cells in vitro and in ex vivo [10,97,100]. Arnoczky et al [10] reported cyclical strain in cultured canine flexor tendon cells induces stress activated protein kinase, which in turn can induce apoptosis [100].…”

“…Oxidative stress can be induced during high dose cyclic strain in human and animal tendon cells in vitro and in ex vivo [10,97,100]. Arnoczky et al [10] reported cyclical strain in cultured canine flexor tendon cells induces stress activated protein kinase, which in turn can induce apoptosis [100]. In a running rat supraspinatus tendon model, we also have found overuse induces upregulation of stress-related genes such as flice inhibitory protein (FLIP), heat shock protein 27 (HSP27) and testis heat shock-related protein 70 (HST70) [75].…”

Cellularity and apoptosis after radiofrequency-induced shrinkage of collagenous tissue

“…Yuan et al (2002) found that 13% of fibroblasts had undergone apoptosis in control sections of the subscapularis tendon. Apoptosis in radiofrequency-treated collagenous tissue at time 0 may occur due to ischemia, hypoxia, free radical generation, heat shock, physical damage or combinations of these factors (Jennings and Reimer 1991, Biagas 1999, Shackelford et al 2000, Skutek et al 2003. The onset of nuclear fragmentation is considered to be relatively short (1-3 h), but the exact onset of apoptosis after thermal treatment remains unclear (Gavrieli et al 1992, Smith et al 2000.…”

“…Although much research has been done, the loading conditions that are neces-sary to improve and accelerate the healing process without causing damage or adverse biomechanical effects remain unclear (Steadman et al 1989, Zeichen et al 2000, Bosch et al 2002, Skutek et al 2003.…”