Selected Research Projects
Overview | Selected Research Projects
Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE2 and LTB4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB4 at 0.1 nM negated the PGE2-induced decrease in cell proliferation. In addition, PGE2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB4 at 0.1 nM. Finally, neither PGE2 nor LTB4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB4 counterbalance the negative effects mediated by PGE2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB4 is generally thought to be pathogenic, low levels of LTB4 are actually beneficial in maintaining tendon tissue homeostasis.
Project description (PDF)
Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.
Project description (PDF)
Fibroblast contraction is necessary for wound closure but excessive contraction contributes to the formation of scar tissue[1]. Two isoforms of transforming growth factor- ß , TGF- ß1 and TGF- ß3, have been shown to induce differential scar tissue formation in vivo [2, 3]. However, the corresponding mechanisms are not clear. Our previous study suggested that TGF- ß1 caused a larger contraction of a population of ligament fibroblasts than TGF- ß3. In this study we used traction force microscopy (TFM) to determine traction forces of individual human patellar tendon fibroblasts (HPTFs) in response to TGF- ß1 and TGF- ß3 treatment.
Project description
Cell traction forces (CTFs) are critical for cell motility and cell shape mainte-
nance. As such, they play a fundamental role in many biological processes such as
angiogenesis, embryogenesis, inflammation, and wound healing. To determine
CTFs at the sub-cellular level with high sensitivity, we have developed high den-
sity micropost force sensor array (MFSA), which consists of an array of vertically
standing poly(dimethylsiloxane) (PDMS) microposts, 2 lm in diameter and 6 lm
in height, with a center-to-center distance of 4 lm. In combination with new
image analysis algorithms, the MFSA can achieve a spatial resolution of 40 nm
and a force sensitivity of 0.5 nN. Culture experiments with various types of cells
showed that this MFSA technology can effectively determine CTFs of cells with
different sizes and traction force magnitudes.
Project description (PDF)
Understanding the relationship between cell shape and cellular function is important
for study of cell biology in general and for regulation of cell phenotype in tissue
engineering in particular. In this study, microcontact printing technique was
used to create cell-adhesive rectangular and circular islands. The rectangular
islands had three aspect ratios: 19.6, 4.9, and 2.2, respectively, whereas circular
islands had a diameter of 50 lm. Both rectangular and circular islands had the
same area of 1960 lm2. In culture, we found that human tendon fibroblasts
(HTFs) assumed the shapes of these islands. Quantitative immunofluorescence
measurement showed that more elongated cells expressed higher collagen type I
than did less stretched cells even though cell spreading area was the same. This
suggests that HTFs, which assume an elongated shape in vivo, have optimal morphology
in terms of expression of collagen type I, which is a major component of
normal tendons. Using immunohistochemistry along with cell traction force
microscopy (CTFM), we further found that these HTFs with different shapes
exhibited variations in actin cytoskeletal structure, spatial arrangement of focal
adhesions, and spatial distribution and magnitude of cell traction forces. The
changes in the actin cytoskeletal structure, focal adhesion distributions, and traction
forces in cells with different shapes may be responsible for altered collagen
expression, as they are known to be involved in cellular mechanotransduction.
Project description (PDF)
Overview | Selected Research Projects
Effect of PGE2 and LTB4 on the proliferation, collagen, and MMP expression of human tendon fibroblasts
Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE2 and LTB4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB4 at 0.1 nM negated the PGE2-induced decrease in cell proliferation. In addition, PGE2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB4 at 0.1 nM. Finally, neither PGE2 nor LTB4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB4 counterbalance the negative effects mediated by PGE2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB4 is generally thought to be pathogenic, low levels of LTB4 are actually beneficial in maintaining tendon tissue homeostasis.Project description (PDF)
EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1beta treatment
Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.Project description (PDF)
Cyclic mechanical stretching modulates IL-1beta induced COX-2 expression and PGE2, production by human tendon fibroblasts
While mechanical loading is known to be essential in maintaining tendon homeostasis, repetitive mechanical loading has also been implicated in the etiology of tendon overuse injuries. The purpose of this study was to determine whether cyclic mechanical stretching regulates inflammatory responses induced by interleukin-1beta (IL-1beta) treatment in human patellar tendon fibroblasts (HPTFs). HPTFs were grown in microgrooved silicone dishes, where they became elongated in shape and aligned with the microgrooves, which is similar to the shape and organization of tendon fibroblasts in vivo. Cyclic uniaxial stretching was then applied to silicone culture dishes with a 4% or 8% stretch at a stretching frequency of 0.5 Hz for a duration of 4 h in the presence or absence of 10 pM IL-1beta treatment. Non-stretched cells in the presence or absence of IL-1beta were used for controls, respectively. The expression of cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1), and the production of prostaglandin E2 (PGE2) were measured. In the absence of stretching, it was found that 10 pM of IL-1beta markedly induced higher levels of COX-2, MMP-1 gene expression, and PGE2 production than non-treated cells. Furthermore, cells with 4% stretching decreased the COX-2 and MMP-1 gene expression and PGE2 production that were stimulated by IL-1beta, whereas cells with 8% stretching further increased these gene products and/or expression levels in addition to the effects of IL-1beta stimulation. Thus, the results suggest that repetitive, small-magnitude stretching is anti-inflammatory, whereas large-magnitude stretching is pro-inflammatory. Therefore, moderate exercise may be beneficial to reducing tendon inflammation.
Project description (PDF)
Project description (PDF)
TGF-beta1 induces greater traction in individual fibroblast than TGF-beta3 — A traction force microscopy study
Fibroblast contraction is necessary for wound closure but excessive contraction contributes to the formation of scar tissue[1]. Two isoforms of transforming growth factor- ß , TGF- ß1 and TGF- ß3, have been shown to induce differential scar tissue formation in vivo [2, 3]. However, the corresponding mechanisms are not clear. Our previous study suggested that TGF- ß1 caused a larger contraction of a population of ligament fibroblasts than TGF- ß3. In this study we used traction force microscopy (TFM) to determine traction forces of individual human patellar tendon fibroblasts (HPTFs) in response to TGF- ß1 and TGF- ß3 treatment.Project description
A micropost force sensor array (MFSA) for traction force study of human fibroblasts
Cell traction forces (CTFs) are critical for cell motility and cell shape mainte-
nance. As such, they play a fundamental role in many biological processes such as
angiogenesis, embryogenesis, inflammation, and wound healing. To determine
CTFs at the sub-cellular level with high sensitivity, we have developed high den-
sity micropost force sensor array (MFSA), which consists of an array of vertically
standing poly(dimethylsiloxane) (PDMS) microposts, 2 lm in diameter and 6 lm
in height, with a center-to-center distance of 4 lm. In combination with new
image analysis algorithms, the MFSA can achieve a spatial resolution of 40 nm
and a force sensitivity of 0.5 nN. Culture experiments with various types of cells
showed that this MFSA technology can effectively determine CTFs of cells with
different sizes and traction force magnitudes.Project description (PDF)
Regulation of cell shape and its effects on cell traction forces and type I collagen expression in human tendon fibroblasts
Understanding the relationship between cell shape and cellular function is important
for study of cell biology in general and for regulation of cell phenotype in tissue
engineering in particular. In this study, microcontact printing technique was
used to create cell-adhesive rectangular and circular islands. The rectangular
islands had three aspect ratios: 19.6, 4.9, and 2.2, respectively, whereas circular
islands had a diameter of 50 lm. Both rectangular and circular islands had the
same area of 1960 lm2. In culture, we found that human tendon fibroblasts
(HTFs) assumed the shapes of these islands. Quantitative immunofluorescence
measurement showed that more elongated cells expressed higher collagen type I
than did less stretched cells even though cell spreading area was the same. This
suggests that HTFs, which assume an elongated shape in vivo, have optimal morphology
in terms of expression of collagen type I, which is a major component of
normal tendons. Using immunohistochemistry along with cell traction force
microscopy (CTFM), we further found that these HTFs with different shapes
exhibited variations in actin cytoskeletal structure, spatial arrangement of focal
adhesions, and spatial distribution and magnitude of cell traction forces. The
changes in the actin cytoskeletal structure, focal adhesion distributions, and traction
forces in cells with different shapes may be responsible for altered collagen
expression, as they are known to be involved in cellular mechanotransduction.Project description (PDF)
Effect of treadmill running on mouse tendons
Characterized by tendon inflammation and/or degeneration, tendinopathy is a serious healthcare
problem in the general population and for athletes as well. While many factors may contribute to
the development of tendinopathy, chronic mechanical loading on tendons is widely believed to
be mainly responsible [Archambault, 1995; Almekinders, 1998; Wang, 2006]. Using “exercise
animal models” to investigate the pathogenic mechanism of tendinopathy [Backman, 1990;
Barbe, 2003; Soslowsky, 2000], previous studies have shown that chronic mechanical loading
causes inflammation and degeneration in tendons. However, the precise pathogenic processes
leading to the development of tendinopathy remain unclear. It is possible that at the early stages
of tendinopathy development, mechanical loading results in tendon microinjuries. Consequently,
the injured tendon undergoes a healing process. Since myofibroblasts are present at the injury
site and are responsible for repairing wound tissues upon injury [Hinz, 2001], we hypothesized
that chronic mechanical loading of tendons through treadmill running induces the presence of
myofibroblasts in mouse tendons.
