scholarly journals P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Rheumatology ◽  
2020 ◽  
Vol 59 (Supplement_2) ◽  
Author(s):  
Linda Lei ◽  
Bahja Abdi ◽  
Amer Mujkavnovic ◽  
Xu Shi Wen ◽  
Henry Lopez ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Collagen Gel ◽  
Explant Culture ◽  
Peptide Inhibitors ◽  
Collagen Gels ◽  
Peripheral Blood Mononuclear ◽  
Relative Expression ◽  
Collagen Mrna ◽  
Collagen Gel Contraction ◽  
New Treatments

Abstract Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.

Beta-adrenergic agonists attenuate fibroblast-mediated contraction of released collagen gels

1996 ◽  
Vol 270 (5) ◽  
pp. L829-L835 ◽  
Author(s):  
T. Mio ◽  
Y. Adachi ◽  
S. Carnevali ◽  
D. J. Romberger ◽  
J. R. Spurzem ◽  
...  
Keyword(s):  
Collagen Gel ◽  
Biologically Active ◽  
Dependent Manner ◽  
Collagen Gels ◽  
Adrenergic Agonists ◽  
Concentration Dependent Manner ◽  
Beta Adrenergic ◽  
Cyclic Monophosphate ◽  
Beta Adrenergic Agonists ◽  
Collagen Gel Contraction

The effects of beta-adrenergic agonists on fibroblast-mediated collagen gel contraction were investigated. beta-Agonists isoproterenol and epinephrine significantly attenuated fibroblast-mediated gel contraction in a concentration-dependent manner, whereas alpha-agonist norepinephrine had no effect. The biologically active form of isoproterenol, (-)-isoproterenol, was 10-fold more effective than the optical isoform, (+)-isoproterenol. beta-Antagonists sotalol and propranolol reversed the attenuation caused by 10(-7) M isoproterenol or epinephrine at the concentration of 10(-7) M or 10(-6) M, but the alpha-antagonist phentolamine did not. However, beta1- or beta2-specificity of these effects is not clear. Isobutyl methylxanthine augmented the effect of isoproterenol and also prolonged the duration. Two reagents which are known to increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP), prostaglandin E2 and dibutyryl adenosine 3',5'-cyclic monophosphate, attenuated gel contraction in a concentration-dependent manner. These data suggest that the fibroblast-mediated collagen gel contraction can be modulated by beta-adrenergic agonists and that the effect depends on cAMP.

Isotonic Biaxial Loading: An Economic, Versatile Method for the Study of Fibroblast-Populated Collagen Gels

1999 ◽  
Author(s):  
Jeffrey W. Holmes ◽  
Thomas K. Borg
Keyword(s):  
Biaxial Loading ◽  
Cardiac Fibroblasts ◽  
Collagen Gel ◽  
Simple System ◽  
Neonatal Rat ◽  
Collagen Gels ◽  
Maximal Contraction ◽  
Contraction Force ◽  
A Value ◽  
Collagen Gel Contraction

Abstract Studies of collagen gel contraction by embedded fibroblasts have characterized the response of free (unloaded) or isometric (maximally loaded) gels but not the response to intermediate loads. An inexpensive, simple system was devised to isotonically load fibroblast-populated collagen gels using freely hanging weights. This system provided excellent repeatability. Maximal contraction force was estimated at 1 mN per million cells in neonatal rat cardiac fibroblasts, a value that agreed well with reported isometric experiments in fibroblasts from other tissues. The ability to load uniaxially or biaxially and with variable loads will facilitate exploration of the regulation of fibroblast mechanics and biology by stress.

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway

2000 ◽  
Vol 278 (5) ◽  
pp. L1032-L1038 ◽  
Author(s):  
X. D. Liu ◽  
C. M. Skold ◽  
T. Umino ◽  
J. R. Spurzem ◽  
D. J. Romberger ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Human Lung ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Lung Fibroblast ◽  
Type I ◽  
Collagen Gels ◽  
Human Lung Fibroblast ◽  
Collagen Gel Contraction ◽  
Cgmp Pathway

Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 μM) significantly augmented collagen gel contraction by HFL1 cells (78.5 ± 0.8 vs. 58.3 ± 2.1, P < 0.01), whereas S-nitroso- N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, NONOate, and N G-monomethyl-l-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1 H-[1,2,4]-oxadiazolo-[4,3- a]-quinoxalin-1-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and PGE2 production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE2 production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE2 production. Addition of exogenous PGE2 blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased PGE2 production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway.

Tetracycline-Based MMP Inhibitors Can Prevent Fibroblast-Mediated Collagen Gel Contraction In Vitro

1998 ◽  
Vol 12 (1) ◽  
pp. 86-93 ◽  
Author(s):  
S.A. Myers ◽  
R.G. Wolowacz
Keyword(s):  
Culture Media ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Cytotoxic Effects ◽  
Chemically Modified ◽  
Tissue Culture Media ◽  
Collagen Gel Contraction ◽  
Chemically Modified Tetracyclines

Collagen gels in vitro can be contracted by fibroblasts. The role of matrix metalloproteinases (MMPs) in the contraction of collagen lattices by human neonatal foreskin fibroblasts (HuFFs) was investigated in tissue culture media supplemented by various doses of known gelatinase inhibitors. Fluorescent assays with model gelatinase substrates and media conditioned by fibroblasts apparently confirmed the ability of chemically modified tetracyclines (CMTs) to act as inhibitors of MMP2, and zymography demonstrated that this was the major cell-derived MMP activity. There were no observable effects on the rate of contraction of attached FPCLs containing 6 x 104 HuFFs (passages 18-25) with either CMT-5 or CMT-2 at all concentrations tested (0-100 μg/mL). However, at greater than 20 μg/mL doxycycline and greater than 5 μg/mL CMT-3, FPCL contraction was completely abolished. Quantitative assessment of cell viability by means of the MTT assay in monolayer and qualitatively within the FPCLs with CalceinAM suggested that differences were not due to cytotoxic effects. Seeding FPCLs with lower-passage fibroblasts produced identical trends. These results may implicate the involvement of MMPs in the process of gel contraction, although tetracyclines have effects additional to their ability to inhibit MMPs directly.

Regulation of fibroblast-mediated collagen gel contraction by platelet-derived growth factor, interleukin-1 alpha and transforming growth factor-beta 1

1992 ◽  
Vol 102 (2) ◽  
pp. 315-322 ◽  
Author(s):  
A. Tingstrom ◽  
C.H. Heldin ◽  
K. Rubin
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Interleukin 1 ◽  
Collagen Gel ◽  
Platelet Derived Growth Factor ◽  
Tgf Beta ◽  
Collagen Gels ◽  
Collagen Gel Contraction ◽  
Growth Factor Beta

We have examined the effects of three macrophage-derived cytokines, platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 alpha (IL-1 alpha) on the contraction of collagen type I gels populated by human foreskin fibroblasts. Contraction was quantified as loss in gel weight. Both PDGF-AA and PDGF-BB were found to induce a rapid collagen-gel contraction. TGF-beta 1 also stimulated gel contraction but with a delayed onset and at a slower rate than the PDGF-stimulated contraction. Rabbit polyclonal IgGs recognizing PDGF-AA and PDGF-BB, respectively, specifically inhibited the effects of the corresponding PDGF isoforms. However, the stimulatory effect of TGF-beta 1 was not affected by any of the anti-PDGF antibodies. The ability of PDGF to stimulate contraction became less pronounced in collagen gel cultures grown in the absence of growth factors over periods of several days. Under the same conditions, the stimulatory effect of TGF-beta 1 was not reduced. The reduced response to PDGF may be due to reduced tension on fibroblasts growing in collagen gels, since fibroblasts on free-floating gels showed a marked reduction in PDGF-BB-induced PDGF beta-receptor aggregates when compared to fibroblasts on attached collagen gels. IL-1 alpha inhibited initial collagen gel contraction, and at later stages induced a visible degradation of the collagen gels, presumably due to the generation of collagenase activity. The combination of IL-1 alpha and PDGF-BB stimulated initial collagen gel contraction, although less effectively than PDGF-BB alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraction of collagen matrices mediated by alpha2beta1A and alpha(v)beta3 integrins

2000 ◽  
Vol 113 (13) ◽  
pp. 2375-2383
Author(s):  
M.E. Cooke ◽  
T. Sakai ◽  
D.F. Mosher
Keyword(s):  
Collagen Gel ◽  
Collagen Gels ◽  
Phosphatidylinositol 3 Kinase ◽  
Fibroblastic Cell ◽  
Collagen Matrices ◽  
Serum Factors ◽  
Stable Transfectants ◽  
Collagen Gel Contraction ◽  
Sphingosine 1 Phosphate ◽  
Alpha V Beta 3

The (beta)1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of (beta)1 and (beta)3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the (beta)1A splice variant of (beta)1 ((beta)1A-GD25) did not express (alpha)2(beta)1A and did not adhere to collagen. After transfection of (alpha)2 into (beta)1A-GD25 cells, the (alpha)2(beta)1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas (beta)1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to (alpha)v(beta)3 or a RGD-containing peptide, indicating that (alpha)v(beta)3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by (alpha)2(beta)1A-GD25 cells was not inhibited by antibodies to (alpha)v(beta)3 or RGD-containing peptide, but was inhibited by anti-(alpha)2 antibody. Flow cytometry demonstrated negligible expression of (alpha)v(beta)3 by (beta)1A-GD25 and (alpha)2(beta)1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by (alpha)2(beta)1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by (alpha)2(beta)1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the (beta)1-null GD25 cells and (beta)1A and (alpha)2(beta)1A transfectants demonstrate that (alpha)2(beta)1A and (alpha)v(beta)3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.

scholarly journals The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure

2002 ◽  
Vol 156 (1) ◽  
pp. 161-172 ◽  
Author(s):  
Jo Ellen Feugate ◽  
QiJing Li ◽  
Lina Wong ◽  
Manuela Martins-Green
Keyword(s):  
Granulation Tissue ◽  
Wound Closure ◽  
Smooth Muscle Actin ◽  
Collagen Gel ◽  
Angiogenic Factor ◽  
Collagen Gels ◽  
Collagen Gel Contraction ◽  
Cxc Chemokine ◽  
Pharmacological Manipulations

Chemokines are small cytokines primarily known for their roles in inflammation. More recently, however, they have been implicated in processes involved in development of the granulation tissue of wounds, but little is known about their functions during this process. Fibroblasts play key roles in this phase of healing: some fibroblasts differentiate into myofibroblasts, α-smooth muscle actin (SMA)-producing cells that are important in wound closure and contraction. Here we show that the CXC chemokine chicken chemotactic and angiogenic factor (cCAF) stimulates fibroblasts to produce high levels of α-SMA and to contract collagen gels more effectively than do normal fibroblasts, both characteristic properties of myofibroblasts. Specific inhibition of α-SMA expression resulted in abrogation of cCAF-induced contraction. Furthermore, application of cCAF to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound closure and contraction. We also show that these effects in culture and in vivo can be achieved by a peptide containing the NH2-terminal 15 amino acids of the cCAF protein and that inhibition of α-SMA expression also results in inhibition of N-peptide–induced collagen gel contraction. We propose that chemokines are major contributors for the differentiation of fibroblasts into myofibroblasts during formation of the repair tissue. Because myofibroblasts are important in many pathological conditions, and because chemokines and their receptors are amenable to pharmacological manipulations, chemokine stimulation of myofibroblast differentiation may have implications for modulation of functions of these cells in vivo.

scholarly journals Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels

2014 ◽  
Vol 462 (1) ◽  
pp. 113-123 ◽  
Author(s):  
Vahid Reyhani ◽  
Pegah Seddigh ◽  
Bengt Guss ◽  
Renata Gustafsson ◽  
Lars Rask ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Collagen Gel ◽  
Protein Binding Site ◽  
Αvβ3 Integrin ◽  
Collagen Gels ◽  
Collagen Gel Contraction ◽  
Native Collagen

The putative functions of extravascular fibrin in pathologies are poorly characterized. We show that fibrinogen binds specifically to a defined protein-binding site of the native collagen. Through this binding, fibrin provides an interface matrix allowing αVβ3 integrin-mediated collagen gel contraction.

Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells

2002 ◽  
Vol 282 (5) ◽  
pp. L1049-L1056 ◽  
Author(s):  
Xiangde Liu ◽  
Tadashi Kohyama ◽  
Hangjun Wang ◽  
Yun Kui Zhu ◽  
Fu-Qiang Wen ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Collagen Gel ◽  
Tissue Remodeling ◽  
Independent Effect ◽  
Airway Smooth Muscle Cells ◽  
Type I ◽  
Dependent Manner ◽  
Th2 Cytokines ◽  
Collagen Gels ◽  
Collagen Gel Contraction

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-β or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) E2 release. Decreased PGE2 release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PGE2release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma.

Molecular and mechano-biology of collagen gel contraction mediated by human MG-63 cells: involvement of specific intracellular signaling pathways and the cytoskeleton

2009 ◽  
Vol 87 (6) ◽  
pp. 895-904 ◽  
Author(s):  
Justin Parreno ◽  
David A. Hart
Keyword(s):  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Potential Role ◽  
Collagen Gel ◽  
Protein Kinase Inhibitors ◽  
Intrinsic Stress ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Collagen Gel Contraction

Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of intrinsic stress. Release of such collagen gels from attachment results in gel contraction and enhanced MMP-1, MMP-3, and α2 integrin mRNA levels. To understand the potential role of microtubules and signaling pathways involved in MG-63 cell-mediated contraction and gene expression, cells were cultured in collagen gels. After 24 h collagen gels were released, then immediately treated with nocodazole or specific protein kinase inhibitors. Contraction was assessed, RNA isolated, and real-time PCR analysis performed. Treatment with high concentrations of a microtubule depolymerization agent, nocodazole, enhanced early contraction and led to elevated mRNA levels for MMP-3, whereas low concentrations inhibited contraction at later time points and did not affect mRNA levels. ROCK inhibitor treatment (Y27632) inhibited collagen gel contraction and led to depressed mRNA levels. The ERK1/2 inhibitor U0126 did not affect contraction, but treatment led to depressed MMP-1, MMP-3, and α2 mRNA levels. The p38MAPK inhibitor SB203580 modestly affected contraction, but did not affect mRNA levels. These results suggest the potential role of cytoskeletal integrity and multiple kinase signaling pathways in specific bone-remodeling events.

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