Disorganized Extracellular Matrix Research Articles

Retinoic acid treatment of human leiomyoma cells transformed the cell phenotype to one strongly resembling myometrial cells

BackgroundUterine leiomyomas are clinically significant tumours that may develop due to an altered differentiation pathway. We have previously identified a dysregulated retinoic acid (RA) pathway that reduced retinoic exposure in human leiomyoma surgical specimens, and have shown that the leiomyoma phenotype was characterized by excessive and disorganized extracellular matrix (ECM).ObjectiveThe goal of this study was to determine the impact of RA exposure on the disrupted ECM phenotype of leiomyomas.Design and methodsStudy of immortalized and molecularly confirmed cells generated from surgical specimens of spontaneous uterine leiomyoma and matched myometrium.ResultsImmortalized leiomyoma and myometrial cells retained the molecular characteristics of their progenitor tissue. Proliferation of leiomyoma cells was inhibited by all-trans retinoic acid (ATRA). Furthermore, there was a dose-dependent decrease in soluble extracellular collagen protein in ATRA-treated leiomyoma cells. Exposure of leiomyoma cells to ATRA resulted in a dose-dependent inhibition of templates for specific ECM protein production including collagen 1, collagen 4, fibronectin and versican. Notably, expression levels in treated leiomyoma cells approached those found in myometrial cells. These mRNA alterations translated into altered protein. Down-regulation was also observed among the RA pathway genes such as CYP26A1 with exposure to ATRA. Finally, ATRA down-regulated TGF-β3 mRNA expression and the TGF-β regulated genes in leiomyoma cells.ConclusionExposure of leiomyomas to ATRA down-regulated cell proliferation, ECM formation, RA metabolism and TGF-β regulation, suggesting that RA exposure can alter the leiomyoma phenotype to one that more closely approximates normal myometrium.

Identification of tissue factor in experimental aortic valve sclerosis

Aortic valve sclerosis (AVS) shares epidemiological and histological similarities with atherosclerosis. Tissue factor (TF), the main initiator of blood coagulation, is present in atherosclerotic plaques and contributes to their thrombogenicity. We aimed to analyze valvular TF expression in addition to other components of atherosclerosis in two models of AVS. Forty-five rabbits were randomly assigned to receive either normal chow (Ctrl, n=15), or 1% cholesterol-enriched chow alone (Hyperchol, n=15) or associated with vitamin D(2) (VitD, n=15), for 12 weeks. Aortic valve (AV) performance, leaflet structure, cellular and lipid infiltration, and TF expression were assessed using Doppler, histology, and immunohistochemistry, respectively, and TF activity was evaluated in AV leaflets. Hyperchol and VitD animals developed abnormal leaflet thickening, with a significant alteration of AV performance in VitD animals. Leaflet thickening was related to the development of fatty plaque neolesions on the aortic side of the leaflets, displaying extracellular matrix disorganization, lipid and cellular infiltration, and calcification in VitD animals. TF was found on the leaflet aortic side in Ctrl animals and was identified in AVS lesions in both Hyperchol and VitD animals. TF immunostaining area and valvular activity increased significantly across the three groups. Experimental AVS lesions that are present on the aortic side of leaflets display numerous characteristics of vascular atherosclerosis, including TF expression. Identification of TF associated with other components of the atherosclerotic process in AVS lesions strengthens the link between atherosclerosis and AVS.

Glycosaminoglycan expression in versican variants is tightly regulated by TGF-β3 in leiomyoma cells

Leiomyomas exhibit a large degree of extracellular matrix (ECM) disorganization. Versican, a hyaluronan-chondroitin sulfate binding glycosaminoglycan (GAG), forms an integral part of the ECM and serves as a repository for growth factors important for leiomyoma proliferation. Versican has four splice variants which express different amounts of GAGs. Versican variant, V0, contains the largest amount of GAGs. Using myometrial and leiomyoma tissue and immortalized cell cultures, we hypothesized that V0 expression would be regulated by TGF-β3, which has been implicated in numerous fibrotic diseases. Laboratory study. After IRB approval, we obtained spontaneous uterine leiomyoma and matched myometrium from hysterectomy specimens. Primary cell cultures were generated and immortalized using recombinant retrovirus containing the E6/E7 open reading frames of HPV type-16. Cells were grown to 80% confluence, serum-starved for 48 hours, and then treated for 24 hours with various concentrations of TGF-β3 and Anti-TGF-β3. V0 expression was analyzed by end point RT-PCR and quantitative RT-PCR in both tissue and immortalized cell cultures. V0 was upregulated in leiomyoma vs. myometrial tissue (mean 3.61, Wilcoxon signed rank P<0.05) and cell culture (mean 2.96, P<0.05). With the addition of TGF-β3, myometrial cells demonstrated increased relative fold expression of V0 compared to untreated myometrial expression (1.65, 34.5, and 11.4, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). Leiomyoma cells exhibited a less pronounced stimulatory effect of TGF-β3 compared to untreated myometrial cells (2.48, 12.9, and 2.62, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). The addition of Anti-TGF-β3 decreased the relative fold expression of leiomyomas cells compared to untreated leiomyomas cells (0.27, 0.20, and 0.02 at 0.1 ug/ml, 1.0 ug/ml, and 10 ug/ml of Anti-TGF-β3). The GAG-rich V0 is overexpressed in leiomyoma vs. myometrial tissue and immortalized cell culture. The addition of TGF-β3 stimulated the expression of V0 in both myometrial and leiomyoma cells, while Anti-TGF-β3 decreased the expression of V0 in leiomyomas cells. Differing levels of GAG composition and amount in leiomyomas contribute to the disorganized ECM by obstructing the tight packing of collagen fibrils. Our data suggest that the larger GAG-rich V0 affects ECM composition and is important to the leiomyoma phenotype.

Knockout Mice: Is It Just Genetics? Effect of Enriched Housing on Fibulin-4+/− Mice

BackgroundFibulin-4 is an extracellular matrix protein expressed by vascular smooth muscle cells that is essential for maintaining arterial integrity. Fibulin-4−/− mice die just before birth due to arterial hemorrhage, but fibulin-4+/− mice appear to be outwardly normal. Experiments were therefore performed to determine whether fibulin-4+/− mice display arterial pathologies on a microscopic scale. After preliminary experiments were performed, a second purpose developed, which was to test the hypothesis that any observed pathologies would be ameliorated by housing the animals in enriched cages.MethodologyFibulin-4+/− and wild-type mice were housed either four/cage in standard cages or two per cage in larger cages, each cage containing a tunnel and a wheel. After three weeks the mice were sacrificed, and the aortas perfusion-fixed and excised for light and electron microscopy.Principle FindingsWhen the mice were in standard cages, localized regions of disorganized extracellular matrix and collagen fibers consistently appeared between some of the medial smooth muscle cells in the fibulin-4+/− mice. In the wild-type mice, the smooth muscle cells were closely connected to each other and the media was more compact. The number of disorganized regions per square mm was significantly greater for fibulin-4+/− mice (172±43 (SEM)) than for wild-type mice (15±8) (p<0.01, n = 8). When the mice were in enriched cages, the fibulin-4+/− mice showed significantly fewer disorganized regions than those in standard cages (35±12) (p<0.05, n = 8). The wild type mice also showed fewer disorganized regions (3±2), but this difference was not significant.ConclusionsThese results indicate that arterial pathologies manifested in fibulin-4+/− mice can be reduced by enriching the housing conditions, and imply that appropriate environments may counteract the effects of some genetic deficiencies.

Effect of enriched housing on fibulin‐4 knockout mice

Fibulin-4 is one of a family of newly recognized extracellular matrix proteins that are expressed by smooth muscle cells and are found in large and small blood vessels. Experiments were performed to test the following hypothesis: arterial pathologies in fibulin-4 knock-out mice are altered by housing conditions. Knock-out mice and wild-type mice were housed four per cage in standard cages, as is the usual practice. After 3 weeks the mice were sacrificed, and the aortas perfusion-fixed and excised for light and electron microscopy. Localized regions of disorganized extracellular matrix and collagen fibers consistently appeared between some of the medial smooth muscle cells in the knockout mice. In the wild-type mice the smooth muscle cells were closely connected to each other and the media was inherently more compact. The number of disorganized regions per 1,000 square microns was significantly greater for knock-out mice (0.172 ± 0.043 (SEM)) than for wild-type mice (0.015 ± 0.008) (p<0.05, n = 8). This study was repeated with the mice housed two per cage in larger cages, each cage containing a tunnel and a wheel. In this case the knock-out mice showed significantly fewer disorganized regions than those in standard cages (0.035 ± 0.012) (p<0.01, n = 8). The wild type mice also showed fewer disorganized regions (0.003 ± 0.002) but this difference was not significant. These results indicate that arterial pathologies manifested in fibulin-4 knock-out mice can be reduced by improving housing conditions, and imply that appropriate environments can counteract the effects of some genetic deficiencies. Supported by NCCAM P20AT00774

Directional Neurite Outgrowth Is Enhanced by Engineered Meningeal Cell-Coated Substrates

After injury to the CNS, the anatomical organization of the tissue is disrupted, posing a barrier to the regeneration of axons. Meningeal cells, a central participant in the CNS tissue response to injury, migrate into the core of the wound site in an unorganized fashion and deposit a disorganized extracellular matrix (ECM) that produces a nonpermissive environment. Previous work in our laboratory has shown that the presentation of nanometer-scale topographic cues to these cells influences their morphological, cytoskeletal, and secreted ECM alignment. In the present study, we provided similar environmental cues to meningeal cells and examined the ability of the composite construct to influence dorsal root ganglion regeneration in vitro. When grown on control surfaces of meningeal cells lacking underlying topographic cues, there was no bias in neurite outgrowth. In contrast, when grown on monolayers of meningeal cells with underlying nanometer-scale topography, neurite outgrowth length was greater and was directed parallel to the underlying surface topography even though there exists an intervening meningeal cell layer. The observed outgrowth was significantly longer than on laminin-coated surfaces, which are considered to be the optimal substrata for promoting outgrowth of dorsal root ganglion neurons in culture. These results suggest that the nanometer-level surface finish of an implanted biomaterial may be used to organize the encapsulation tissue that accompanies the implantation of materials into the CNS. It furthermore suggests a simple approach for improving bridging materials for repair of nerve tracts or for affecting cellular organization at a device-tissue interface.

Regulation of the collagen phenotype expression of gamma-irradiated vascular smooth muscle cells by heparan mimetics (RGTA).

Restenosis is characterized by vascular smooth muscle cell (VSMC) proliferation and accumulation of collagen III in a hypertrophic and disorganized extracellular matrix. Restenosis is prevented by antimitotic agents or irradiation but no significant progress has been made to control collagen expression deregulation. Previously, we have shown that a new family of biopolymers named RGTA (heparan mimetics elaborated by grafting on dextran of carboxylate, sulfate, and benzylamide units) stimulate in vivo tissue repair and reduce fibrosis in various models. Using VSMC in vitro (pig aortic VSMC irradiated with a 60Co source and labeled with [3H]Proline), we now show that gamma-irradiation reduced cell survival by 50% and collagen synthesis 6-fold with a major increase in the ratio of collagen III to collagen I biosynthesis taken as a fibrotic index. RGTA added to the cells enhanced their survival up to 80% and reduced collagen III/I ratio back to values found in normal vascular tissues. These results suggest that RGTA combined with gamma-radiation could be an efficient strategy against restenosis.

The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox.

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many region-specific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the 'flagellar hillocks' that encircle the basal ends of all flagella, and in the adjacent 'boundary zone' that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.

Skin elastic fibers in Williams syndrome

The elastin gene is consistently deleted in Williams syndrome and as this protein represents the major component of the elastic fibers of the dermis, we sought to investigate skin elastic fibers in Williams syndrome as a key to unraveling extracellular matrix disorganization in this condition. Both morphometric parameters analyzed by using automated image analysis and immunofluorescence labeling with monoclonal antibodies against elastin and fibrillin 1 showed a disorganized pre-elastic (oxytalan and elaunin) and mature elastic fibers in the dermis of 10 Williams syndrome patients compared with five healthy children and one patient with isolated supravalvular aortic stenosis. Skin biopsies in Williams syndrome patients provide a simple mean to elucidate extracellular matrix anomalies. Hopefully, this method could give clues to the understanding of the elastic network anomalies in this condition and even to the consequences of these latter on elasticity and resilience of other tissues such as the arterial tree. Am. J. Med. Genet. 87:134–138, 1999. © 1999 Wiley-Liss, Inc.

Skin elastic fibers in Williams syndrome

The elastin gene is consistently deleted in Williams syndrome and as this protein represents the major component of the elastic fibers of the dermis, we sought to investigate skin elastic fibers in Williams syndrome as a key to unraveling extracellular matrix disorganization in this condition. Both morphometric parameters analyzed by using automated image analysis and immunofluorescence labeling with monoclonal antibodies against elastin and fibrillin 1 showed a disorganized pre-elastic (oxytalan and elaunin) and mature elastic fibers in the dermis of 10 Williams syndrome patients compared with five healthy children and one patient with isolated supravalvular aortic stenosis. Skin biopsies in Williams syndrome patients provide a simple mean to elucidate extracellular matrix anomalies. Hopefully, this method could give clues to the understanding of the elastic network anomalies in this condition and even to the consequences of these latter on elasticity and resilience of other tissues such as the arterial tree.

Effect of Tenascin-C Deficiency on Chemically Induced Dermatitis in the Mouse

Tenascin-C is a large extracellular matrix glycoprotein characterized by its spatiotemporal expression during embryogenesis, carcinogenesis, and wound healing. Many in vitro studies on tenascin-C have revealed its multifunctional properties; however, disruption of the tenascin-C gene did not reveal any obvious abnormalities during development, and its function in vivo remains unclear. Here, we investigated whether tenascin-C is involved in inflammatory dermatitis in adults by studying chemically induced dermatitis in tenascin-C knockout mice. An epicutaneous application of a hapten, 2,4-dinitrofluorobenzene, to the ear skin of BALB/CA mice resulted in inflammation and induced the expression of tenascin-C. In congenic tenascin-C knockout mice, the dermatitis occurred more severely than in wild-type mice; infiltration of polymorphonuclear cells in knockout mice persisted longer than in wild-type mice, and the elastosis-like disorganized extracellular matrix was also seen in the ear. These results suggest that tenascin-C plays a role in vivo in inflammatory responses in the skin, and that the genetic background has profound effects on the function of tenascin-C in mouse dermatitis.

Type X collagen alterations in rachitic chick epiphyseal growth cartilage.

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.