Cyclic mechanical strain induces TGFβ1-signalling in dermal fibroblasts embedded in a 3D collagen lattice

We reported previously that, in addition to mechanical strain, a constrained boundary condition alone can alter the organization of microvessel outgrowth during in vitro angiogenesis [1]. After 6 days of culture in vitro, microvessels aligned parallel to the long axis of rectangular 3D collagen gels that had constrained edges on the ends. However, unconstrained cultures did not show any alignment of microvessels. The ability to direct microvessel outgrowth during angiogenesis has significant implications for engineering prevascularized grafts and tissues in vitro, therefore an understanding of this process is important. Since there is direct relationship between the ability of endothelial cells to contract 3D gels and matrix stiffness [2], we hypothesize that some constrained boundary conditions will increase the apparent matrix stiffness and in turn will limit gel contraction, prevent microvessel alignment, and reduce microvessel outgrowth. The objective of this study was to compare microvessel growth and alignment under several different static boundary conditions.

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Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices

Gels ◽

10.3390/gels6040033 ◽

2020 ◽

Vol 6 (4) ◽

pp. 33

Author(s):

Jiranuwat Sapudom ◽

Claudia Damaris Müller ◽

Khiet-Tam Nguyen ◽

Steve Martin ◽

Ulf Anderegg ◽

...

Keyword(s):

Extracellular Matrix ◽

Tumor Microenvironment ◽

Average Molecular Weight ◽

Cell Types ◽

Dermal Fibroblasts ◽

Matrix Remodeling ◽

Cancer Associated Fibroblasts ◽

Collagen Matrices ◽

3D Collagen ◽

Tgf Β1

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

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Mechanical stimulation of human dermal fibroblasts regulates pro-inflammatory cytokines: potential insight into soft tissue manual therapies

10.21203/rs.2.18427/v2 ◽

2020 ◽

Author(s):

Aric Anloague ◽

Aaron Mahoney ◽

Oladipupo Ogunbekun ◽

William R. Thompson ◽

Bryan Larsen ◽

...

Keyword(s):

Soft Tissue ◽

Inflammatory Cytokines ◽

Mechanical Stimulation ◽

Mechanical Strain ◽

Dermal Fibroblasts ◽

Manual Therapies ◽

Human Dermal Fibroblasts ◽

Stimulation Of ◽

Pro Inflammatory Cytokines

Abstract Objective Soft tissue manual therapies are commonly utilized by osteopathic physicians, chiropractors, physical therapists and massage therapists. These techniques are predicated on subjecting tissues to biophysical mechanical stimulation but the cellular and molecular mechanism(s) mediating these effects are poorly understood. A series of previous studies established an in vitro model system for examining mechanical stimulation of dermal fibroblasts and established that repetitive strain, intended to mimic overuse injury, induces the secretion of numerous pro-inflammatory cytokines. Moreover, mechanical strain intended to mimic soft tissue manual therapy reduces strain-induced secretion of pro-inflammatory cytokines. Here, we sought to partially confirm and extend these reports and provide independent corroboration of prior results. Results Using cultures of primary human dermal fibroblasts, we confirm mechanical forces intended to mimic repetitive motion strain increases levels of IL-6 and that mechanical strain intended to mimic therapeutic soft tissue stimulation reduces IL-6 levels. We also extend the prior work, reporting that therapy-like mechanical stimulation reduces levels of IL-8. Although there are important limitations to this experimental model, these findings provide supportive evidence that therapeutic soft tissue massage may reduce inflammation. Future work is required to address these open questions and advance the mechanistic understanding of therapeutic soft tissue stimulation.

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An Aqueous Extract of the Leaves ofChromolaena odorata(FormerlyEupatorium odoratum) (Eupolin) Inhibits Hydrated Collagen Lattice Contraction by Normal Human Dermal Fibroblasts

The Journal of Alternative and Complementary Medicine ◽

10.1089/acm.1996.2.335 ◽

1996 ◽

Vol 2 (3) ◽

pp. 335-343 ◽

Cited By ~ 23

Author(s):

Thang T. Phan ◽

Margaret A. Hughes ◽

George W. Cherry ◽

Trung T. Le ◽

Hung M. Pham

Keyword(s):

Aqueous Extract ◽

Dermal Fibroblasts ◽

Human Dermal Fibroblasts ◽

Lattice Contraction ◽

Collagen Lattice ◽

Normal Human ◽

Normal Human Dermal Fibroblasts

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Feedback Inhibition of High TGF-β1 Concentrations on Myofibroblast Induction and Contraction by Dupuytren’s Fibroblasts

Journal of Hand Surgery (European Volume) ◽

10.1016/j.jhsb.2006.05.007 ◽

2006 ◽

Vol 31 (5) ◽

pp. 473-483 ◽

Cited By ~ 15

Author(s):

M. WONG ◽

V. MUDERA

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Feedback Inhibition ◽

Transforming Growth Factor Β1 ◽

Dermal Fibroblasts ◽

Flexor Retinaculum ◽

3D Collagen ◽

Contractile Forces ◽

Higher Doses ◽

Tgf Β1

Myofibroblasts and TGF-β1 are implicated in Dupuytren’s contracture. Transforming growth factor β1(TGF-β1) (1–10 ng/ml) increases myofibroblast induction in Dupuytren’s fibroblasts and contraction in a collagen model. However, higher doses (20–30 ng/ml) inhibit contraction in dermal fibroblasts. We hypothesized higher doses of TGF-β1 would inhibit induction of myofibroblasts and contraction by Dupuytren’s fibroblasts. Increasing doses of TGF-β1 (0–30 ng/ml) were tested on Dupuytren’s fibroblasts using immunofluorescence to determine myofibroblast upregulation and a 3D collagen model used to determine contractile forces. Flexor retinaculum fibroblasts were used as controls. TGF-β1 induced myofibroblasts in Dupuytren’s fibroblasts ( n = 3) from 12% (0 ng/ml) to 23% (12.5 ng/ml) at 24 hours but dropped to 13% at 30 ng/ml ( P < 0.05). This response was mirrored in the contraction profiles. These trends were similar for flexor retinaculum fibroblasts ( n = 3), but contractile forces and myofibroblast induction were significantly less ( P < 0.001). This is the first report of negative feedback inhibition of TGF-β1 at higher concentrations in Dupuytren’s fibroblasts.

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Contracted capsule fibroblast conditioned media induces increased contraction of a normal breast tissue derived fibroblast-populated 3D collagen lattice via paracrine signalling

Journal of Plastic Reconstructive & Aesthetic Surgery ◽

10.1016/j.bjps.2014.08.019 ◽

2014 ◽

Vol 67 (10) ◽

pp. 1461

Author(s):

Daniel J. Kyle ◽

Ardeshir Bayat

Keyword(s):

Breast Tissue ◽

Normal Breast Tissue ◽

Normal Breast ◽

Conditioned Media ◽

Paracrine Signalling ◽

Collagen Lattice ◽

3D Collagen

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FACTOR XIII (FXIIIa) OF BLOOD COAGULATION NORMALIZES COLLAGEN SYNTHESIS OF SCLERODERMA FIBROBLASTS

10.1055/s-0038-1644650 ◽

1987 ◽

Author(s):

M Paye ◽

T Krieg ◽

Ch M Lapière

Keyword(s):

Collagen Synthesis ◽

Normal Skin ◽

Culture Conditions ◽

Dermal Fibroblasts ◽

Skin Fibroblasts ◽

Similar Proportion ◽

Collagen Biosynthesis ◽

Active Lesion ◽

Collagen Production ◽

Collagen Lattice

Progressive systemic scleroderma (PSS) fibroblasts display, in some cases, an excessive collagen production which leads to fibrosis of the skin and internal organs. Administration of FXIIIa has been reported to be beneficial to some of the PSS patients. The effect of FXIIIa on collagen synthesis by PSS fibroblasts was studied in vitro in two different culture conditions: in a confluent monolayer on plastic and in a three-dimensional collagen lattice.Proteins and collagen synthesis was measurfed by metabolic labeling for 24 h with 3H-proline in absence or presence of FXIIIa (1 U/ml) in dermal fibroblasts from an active lesion (PSS forearm), from an uninvolved area of the skin of the same patient (control abdomen) and from the skin of a normal subject.Proteins synthesis was similar for the three strains under both culture conditions while collagen synthesis was strongly increased in PSS forearm fibroblasts as compared to the uninvolved and to the normal skin fibroblasts. The addition of FXIIIa repressed collagen synthesis of PSS forearm synthesis to the level observed in the abdominal skin fibroblasts of the patient and the normal cells in absence of FXIIIa. When cultured in a collagen lattice collagen synthesis was repressed in a similar proportion in all fibroblasts. Addition of FXIIIa further reduced collagen biosynthesis in the active PSS fibroblasts. The addition of FXIIIa in the lattice largely increased the degradation of newly synthesized collagen in all the strains of fibroblasts.The action of FXIIIa on collagen biosynthesis was also tested in monolayer in 3 other strains of PSS fibroblasts and 3 controls. In all fibroblasts, collagen biosynthesis was reduced by 75 % after addition of FXIIIa.Our results suggest that the beneficial effect of FXIIIa administration in PSS patient might be related to a reduction o1 excessive collagen production and perhaps to an increased degradation of newly synthesized molecules.

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Mechanical stimulation of human dermal fibroblasts regulates pro-inflammatory cytokines: potential insight into soft tissue manual therapies

10.21203/rs.2.18427/v3 ◽

2020 ◽

Author(s):

Aric Anloague ◽

Aaron Mahoney ◽

Oladipupo Ogunbekun ◽

Taylor Hiland ◽

William R. Thompson ◽

...

Keyword(s):

Soft Tissue ◽

Inflammatory Cytokines ◽

Mechanical Stimulation ◽

Mechanical Strain ◽

Dermal Fibroblasts ◽

Manual Therapies ◽

Human Dermal Fibroblasts ◽

Long Duration ◽

Stimulation Of ◽

Pro Inflammatory Cytokines

Abstract ObjectiveSoft tissue manual therapies are commonly utilized by osteopathic physicians, chiropractors, physical therapists and massage therapists. These techniques are predicated on subjecting tissues to biophysical mechanical stimulation but the cellular and molecular mechanism(s) mediating these effects are poorly understood. Previous studies established an in vitro model system for examining mechanical stimulation of dermal fibroblasts and established that cyclical strain, intended to mimic overuse injury, induces secretion of numerous pro-inflammatory cytokines. Moreover, mechanical strain intended to mimic soft tissue manual therapy reduces strain-induced secretion of pro-inflammatory cytokines. Here, we sought to partially confirm and extend these reports and provide independent corroboration of prior results.ResultsUsing cultures of primary human dermal fibroblasts, we confirm cyclical mechanical strain increases levels of IL-6 and adding long-duration stretch, intended to mimic therapeutic soft tissue stimulation, after cyclical strain results in lower IL-6 levels. We also extend the prior work, reporting that long-duration stretch results in lower levels of IL-8. Although there are important limitations to this experimental model, these findings provide supportive evidence that therapeutic soft tissue stimulation may reduce levels of pro-inflammatory cytokines. Future work is required to address these open questions and advance the mechanistic understanding of therapeutic soft tissue stimulation.

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A Three-dimensional Collagen Lattice Induces Protein Kinase C-ζ Activity: Role in α2 Integrin and Collagenase mRNA Expression

The Journal of Cell Biology ◽

10.1083/jcb.136.2.473 ◽

1997 ◽

Vol 136 (2) ◽

pp. 473-483 ◽

Cited By ~ 75

Author(s):

Jiahua Xu ◽

Richard A.F. Clark

Keyword(s):

Protein Kinase C ◽

Mrna Expression ◽

Three Dimensional ◽

Collagen Gel ◽

Collagen Matrix ◽

Dermal Fibroblasts ◽

Kinase C ◽

Collagen Lattice ◽

Pkc Ζ ◽

Collagen Receptor

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor α2 subunit mRNA expression in both unstimulated or platelet-derived growth factor–stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239–249). Here we report that the collagen gel can signal protein kinase C (PKC)-ζ activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-ζ immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-ζ protein levels or intracellular location was observed. DNA binding activity of nuclear factor κB (NF-κB), a downstream regulatory target of PKC-ζ, was also increased by fibroblasts grown in collagen gel. The composition of the NF-κB/Rel complexes that contained p50, was not changed. The potential role of PKC-ζ in collagen gel–induced mRNA expression of collagen receptor α2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of α2 and MMP-1 mRNA in collagen gel–stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)–inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5′ end of PKC-ζ mRNA sequences significantly reduced the collagen lattice–stimulated α2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-ζ, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for α2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-ζ.

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