GABA receptors and nitric oxide ameliorate constrictive collagen remodeling in hyperhomocysteinemia

Elevated plasma levels of homocysteine (Hcy) are associated with vascular dementias and Alzheimer's disease. The role of Hcy in brain microvascular endothelial cell (MVEC) remodeling is unclear. Hcy competes with muscimol, an gamma-amino butyric acid (GABA)-A receptor agonist. GABA is the primary inhibitory neurotransmitter in the brain. Our hypothesis is that Hcy induces constrictive microvascular remodeling by altering GABA-A/B receptors. MVEC from wild type, matrix metalloproteinase-9 (MMP-9) knockout (-/-), heterozygote cystathionine beta synthase (CBS-/+), and endothelial nitric oxide synthase knockout (eNOS-/-) mouse brains were isolated. The MVEC were incorporated into collagen (3.2 mg/ml) gels and the decrease in collagen gel diameter at 24 h was used as an index of constrictive MVEC remodeling. Gels in the absence or presence of Hcy were incubated with muscimol or baclofen, a GABA-B receptor agonist. The results suggested that Hcy-mediated MVEC collagen gel constriction was ameliorated by muscimol, baclofen, MMP-9, and eNOS gene ablations. There was no effect of anti-alpha 3 integrin. However, Hcy-mediated brain MVEC collagen constriction was abrogated with anti-beta-1 integrin. The co-incubation of Hcy with L-arginine ameliorated the Hcy-mediated collagen gel constriction. The results of this study indicated amelioration of Hcy-induced MVEC collagen gel constriction by induction of nitric oxide through GABA-A and -B receptors.     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

The behaviour of BHK cells and neutrophil leukocytes on collagen gels of defined mechanical strength

This study demonstrates how the mechanical strength of a series of collagen/composite gels can be measured using a penetrometer. It was found that the presence of fibrin in collagen gels resulted in increased gel strength. Similarly hyaluronic acid was found to increase the strength of collagen gels. Addition of heparin weakened collagen gels as did chondroitin-6-sulphate. Neutrophil migration into collagen gels was found to be inversely proportional to gel strength. Fibrin and hyaluronic acid containing gels inhibited neutrophil migration while the presence of heparin and chondroitin sulphate increased neutrophil migration. BHK gel contraction experiments demonstrated how the presence of fibrin prevents gel contraction. Despite increasing gel strength the presence of hyaluronic acid appeared to have no effect on BHK contraction of collagen gels. Similarly the presence of heparin or chondroitin sulphate had no effect on gel contraction by BHK cells.     

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-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 microM) 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 1H-[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 PGE(2) production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE(2) production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE(2) production. Addition of exogenous PGE(2) 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 PGE(2) 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.     

The effect of coculture with human smooth muscle cells on the proliferation,the IL‐1 β secretion,the PDGF production and tube formation of human aortic endothelial cells

Endothelial cells (ECs) and smooth muscle cells (SMCs), which are the major component cells of blood vessels, produce various bioactive substances and communicate with each other through them. Although several studies of the interaction between ECs and SMCs have been reported, the effect of coculture with SMCs on ECs is still obscure. To clarify the interaction of ECs and SMCs, we examined the effect of coculture with SMCs on the proliferation, the IL‐1β secretion, the PDGF production and tube formation of ECs, using the coculture model: transferable wells and collagen gel. IL‐1 and PDGF are considered to be related to progression of atherosclerosis. Proliferation and tube formation of ECs are associated with repair of vessels. In the transferable well system coculture with SMCs stimulated the proliferation of ECs, and enhanced the IL‐1β secretion of ECs and in the collagen gel system coculture with SMCs induced the tube formation of ECs, and appeared to enhance the PDGF production of ECs. In conclusion, the effect of coculture with SMCs on ECs has two conflicting aspects: progression of atherosclerosis and angiogenesis. These results suggest that an imbalance of their effects may lead to pathological events. Copyright © 1999 John Wiley & Sons, Ltd.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

Improved arterial wall model by coculturing vascular endothelial and smooth muscle cells

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.     

Synergistic effect of 4-hydroxynonenal and PPAR ligands in controlling human leukemic cell growth and differentiation

Peroxisome proliferator-activated receptors play an important role in the differentiation of different cell lines. In this study we demonstrate that PPAR-alpha ligands (clofibrate and ciprofibrate) and PPAR-gamma ligands (troglitazone and 15d-prostaglandin J2) inhibit growth and induce monocytic differentiation in HL-60 cells, whereas only PPAR-gamma ligands inhibit growth of U937 cells. Differentiation was demonstrated by the analysis of surface antigen expression CD11b and CD14, and by the characteristic morphological changes. PPAR-gamma ligands are more effective than PPAR-alpha ligands in the inhibition of cell growth and in the induction of differentiation. The physiological product of lipid peroxidation, 4-hydroxynonenal (HNE), which alone induces granulocytic-like differentiation of HL-60 cells, potentiates the monocytic differentiation induced by ciprofibrate, troglitazone, and 15d-prostaglandin J2. The same HNE treatment significantly inhibits U937 cell growth and potentiates the inhibition of cell growth in PPAR-gamma ligand-treated cells. However, HNE does not induce a significant number of CD14-positive U937 cells. HNE causes a great increase of PPAR-gamma expression in both HL-60 and U937 cells, whereas it does not modify the PPAR-alpha expression. This observation may account for the high synergistic effect displayed by HNE and PPAR-gamma ligands in the inhibition of cell growth and differentiation induction. These results represent the first evidence of the involvement of a product of lipid peroxidation in the modulation of PPAR ligand activity and suggest a relationship between HNE and PPAR ligand pathways in leukemic cell growth and differentiation.     

15-Deoxy-Delta(12,14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells.

Apoptosis has been described in placental (trophoblast) tissues during both normal and abnormal pregnancies. We have studied the effects of the cyclopentenone prostaglandins (PGs) on trophoblast cell death using JEG3 choriocarcinoma cells. PGJ(2), Delta(12)PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)) (10 microM) significantly reduced mitochondrial activity (MTT assay) over 16 h by 17.4 +/- 4.7%, 28 +/- 9.3%, and 62.5 +/- 2.8%, respectively (mean +/- sem), while PGA(2) and PGD(2) had no effect. The synthetic PPAR-gamma ligand ciglitizone (12.5 microM) had a potency similar to 15dPGJ(2) (69 +/- 3% reduction). Morphological examination of cultures treated with PGJ(2) and its derivatives revealed the presence of numerous cells with dense, pyknotic nuclei, a hallmark of apoptosis. FACS analysis revealed an abundance (approximately 40%) of apoptotic cells after 16-h treatment with 15dPGJ(2) (10 microM). The caspase inhibitor ZVAD-fmk (5 microM) significantly diminished the apoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2). JEG3 cells expressed PPAR-gamma mRNA by Northern analysis. These novel findings imply a role for PPAR-gamma ligands in various processes associated with pregnancy and parturition.     

Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.     

Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE(2), PGA(2), PGA(1), PGF(2 alpha)) either delayed apoptosis or had no effect, whereas PGD(2) and its metabolite PGJ(2) selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca(2+). Intriguingly, the sequential metabolites Delta(12)PGJ(2) and 15-deoxy-Delta(12,) Delta(14)-PGJ(2) (15dPGJ(2)) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Delta(12)PGJ(2) and 15dPGJ(2) are peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, apoptosis was not mimicked by synthetic PPAR-gamma and PPAR-alpha ligands or blocked by an irreversible PPAR-gamma antagonist. Furthermore, Delta(12)PGJ(2) and 15dPGJ(2) inhibited LPS-induced I kappa B alpha degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-gamma-independent inhibition of NF-kappa B activation. In addition, we show that TNF-alpha-mediated loss of cytoplasmic I kappa B alpha in eosinophils is inhibited by 15dPGJ(2) in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD(2) and PGJ(2) may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2) in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.     

Mechanisms of stiffening and strengthening in media-equivalents fabricated using glycation

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.     

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).     

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.     

Novel spectroscopic technique for in situ monitoring of collagen fibril alignment in gels

Development of collagen fibril alignment in contracting fibroblast-populated and externally tensioned acellular collagen gels was studied using elastic scattering spectroscopy. Spectra of the backscattered light (320-860 nm) were acquired with a 2.75-mm source-detector separation probe placed perpendicular to the gel surface and rotated to achieve different angles to the collagen fibril alignment. Backscatter was isotropic for noncontracted/unloaded gels (disorganized matrix). As gels were contracted/externally loaded (collagen alignment developed), anisotropy of backscatter increased: more backscatter was detected perpendicular than parallel to the direction of the fibril alignment. An "anisotropy factor" (AF) was calculated to characterize this effect as the ratio of backscatter intensities at orthogonal positions. Before contraction (or zero strain) the AF was close to unity at all wavelengths. In contrast, at 72 h, backscatter anisotropy varied from AF(400 nm) = 2.14 +/- 0.29 to AF(700 nm) = 3.04 +/- 0.48. It also increased over threefold up to a strain of 20%. The AF strongly correlated with the contraction time/strain. Different directions of the backscatter were detected in gel zones with known differences in the matrix alignment. Thus, backscatter anisotropy allows in situ nondestructive determination of collagen fibril alignment and quantitative monitoring of its development.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

An improved method for the collagen gel contraction assay

The collagen gel contraction (CGC) assay is used frequently to study the cell-mediated reorganization of the extracellular natrix. In a typical CGC assay, cells embedded in a disk-shaped lattice (gel) of native type I collagen fibers compress the fibers and, consequently, reduce the diameter of the collagen disk within h or d. The degree to which the collagen is contracted is usually quantified by measurement of the diameter or the area of the disk. During CCC assays, friction or adhesion (or both) between gels and their culture containers can cause gels to be incompletely contracted or to acquire distorted shapes. Such occurrences degrade the reproducibility and reliability of measurements of gel dimensions. To address these problems, we developed an oil-supported collagen retraction (OSCR) assay that creates an environment of low friction and adhesion around the contracting collagen gel. The OSCR assay is accomplished with simple equipment and is easily performed, sensitive, and consistently yields fully contracted gels with minimal distortion.