Developmentally regulated changes in extracellular matrix in endothelial and smooth muscle cells in the ductus arteriosus may be related to intimal proliferation

In the late gestation fetal lamb ductus arteriosus (DA), intimal proliferation is observed, characterized by smooth muscle migration and proliferation in the subendothelium. The nature of changes in the endothelial and smooth muscle extracellular matrix associated with the development of this feature are not known. We assessed the production of glycoproteins (fibronectin, laminin, and type IV collagen) and glycosaminoglycans (GAGs) (hyaluronic acid, heparan sulfate, and chondroitin sulfate) in endothelial and smooth muscle cells harvested from the DA, aorta (Ao), and pulmonary artery of fetal lambs at 100 days gestation, before the appearance of DA intimal proliferation, and at 138 days, when well-developed intimal 'cushions' are seen. In passage 3 cells, glycoprotein synthesis was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after 48 hours incubation with [35S]methionine, and GAGs were assessed by labeling with [3H] glucosamine and separation on DEAE ion-exchange high performance liquid chromatography. Analyses were carried out on culture medium, cell layer, and solubilized matrix. Fibronectin secretion by DA smooth muscle cells from 100-day lambs was found to be twice that of Ao or pulmonary artery cells. No significant differences were seen in smooth muscle cells from 138-day lambs or when comparing endothelial cells from each of the vascular sites at both gestational ages. As well, there were no DA-specific differences in laminin or type IV collagen. No significant differences in endothelial GAG secretion were observed comparing each vascular site at both gestational ages. Analysis of endothelial-derived matrices, however, revealed increased incorporation of hyaluronic acid in the DA from 100-day lambs, 10-fold that of the pulmonary artery and Ao, and increased heparan sulfate. These differences were still present in cell matrices from late gestation animals, but were less marked. No differences in GAGs were seen when comparing smooth muscle cells. Incubation of 100-day DA and Ao smooth muscle cells with endothelial conditioned medium however, resulted in a 2-fold increase in chondroitin sulfate in DA, compared with Ao. These results indicate that distinct, developmentally regulated patterns of extracellular matrix production are related to vascular site and specific features appear to precede intimal proliferation in the DA.     

Hyaluronic acid accumulation and endothelial cell detachment in intimal thickening of the vessel wall. The normal and genetically defective ductus arteriosus.

The closing ductus arteriosus (DA) was studied as a model for the development of intimal thickening of vessel walls using ultrastructural and immunohistochemical techniques. The material consisted of DA from neonatal dogs of three types: normal beagles, DA-defective pups from a line of mixed poodles with a genetic defect in the closure of the DA leading to persistent ductus arteriosus (PDA line), and normal litter-mates of DA-defective pups in the PDA line. The DA of the normal litter-mates of DA-defective pups did not differ from those of normal beagles. In the DA of normal beagles and normal PDA-line pups, closure is preceded by intimal thickening characterized by formation of a widened subendothelial region (SR), detachment of endothelial cells, invagination of endothelial cells, and migration of smooth muscle cells into the SR. It was observed that immediately before and after endothelial cell detachment, there was an increase in hyaluronic acid (HA) in the SR and inner media. In the DA-defective pups, the increase in hyaluronic acid failed to occur and there was no intimal thickening. The SR failed to expand, endothelium remained attached to the internal elastic membrane, and there was no invagination of endothelium or migration of smooth muscle cells. It is hypothesized that the increased synthesis of HA is an important early event leading to intimal thickening in the normal DA and perhaps to abnormal intimal thickening of other vessels. By its hygroscopic properties, HA may be directly involved in the formation of a wide SR, inducing endothelial cell detachment and favoring smooth muscle cell migration. In affected pups of the PDA line, there is a genetically-determined "block" in the normal process of intimal thickening at or before the initiation of increased HA synthesis.     

Characterization of myxoid substance of human aortic sarcoma

An autopsy case of aortic sarcoma which was located in the intima of the abdominal aorta and characterized by abundant extracellular myxoid substances was examined. Histological, immunohistochemical and ultrastructural studies revealed no characteristics suggestive of endothelial, leiomyogenic or histiocytic differentiation. Histochemical and biochemical analyses revealed that the myxoid substances were glycosaminoglycans (GAGs) consisting of hyaluronic acid (85% of the total GAG) and chondroitin sulfate proteoglycan (15% of the total GAG). Neither heparan sulfate proteoglycan nor dermatan sulfate proteoglycan was detected. In the aorta, GAGs are synthesized mainly by arterial smooth muscle cells and endothelial cells. Among these cells, the arterial smooth muscle cell is the principal cell type that synthesizes aortic GAGs, predominantly chondroitin sulfate proteoglycan, whereas the endothelial cell synthesizes small amounts of GAGs, predominantly heparan sulfate proteoglycan. Therefore, the results of this study suggest that the tumor cells of the present case have a property similar to arterial smooth muscle cells in terms of GAG synthesis, and that the tumor originated most probably from arterial smooth muscle cells.     

Characterization of Myxoid Substance of Human Aortic Sarcoma

An autopsy case of aortic sarcoma which was located in the intima of the abdominal aorta and characterized by abundant extracellular myxoid substances was examined. Histological, immunohistochemical and ultrastructural studies revealed no characteristics suggestive of endothelial, leiomyogenic or histiocytic differentiation. Histochemical and biochemical analyses revealed that the myxoid substances were glycosaminoglycans (GAGs) consisting of hyaluronic acid (85% of the total GAG) and chondroitin sulfate proteoglycan (15% of the total GAG). Neither heparan sulfate proteoglycan nor dermatan sulfate proteoglycan was detected. In the aorta, GAGs are synthesized mainly by arterial smooth muscle cells and endothelial cells. Among these cells, the arterial smooth muscle cell is the principal cell type that synthesizes aortic GAGs, predominantly chondroitin sulfate proteoglycan, whereas the endothelial cell synthesizes small amounts of GAGs, predominantly heparan sulfate proteoglycan. Therefore, the results of this study suggest that the tumor cells of the present case have a property similar to arterial smooth muscle cells in terms of GAG synthesis, and that the tumor originated most probably from arterial smooth muscle cells.     

Fibronectin isoforms in megakaryocytes

Our studies have shown that megakaryocytes (MK) can synthesize fibronectin (FN) and alternatively spliced fibronectin, FN EIIIB. FN EIIIB is primarily present in embryonic, proliferating and migrating cells, and thought to be important for cell maturation. MK, but not nonmegakaryocytic bone marrow cells, contain FN EIIIB and thus, MK and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. Thrombin can induce the secretion of general FN, but does not cause the secretion of FN EIIIB into the medium. Analysis of immunostained cells by confocal microscopy revealed that both general FN and FN EIIIB accumulated on the MK surface following thrombin treatment. Thus, FN EIIIB can be released only to be bound to the MK surface. The expression of FN EIIIB on the MK surface may have a unique role in MK migration and maturation.     

Effects of aldosterone on synthesis of fibronectin and expression of transforming growth factor-beta1 mRNA in cultured rat mesangial cells]

AIM: To evaluate the effect of aldosterone (ALD) and spironolactone (SPI, one of the aldosterone receptor antagonists) on synthesis and secretion of fibronectin (FN) and expression of transforming growth factor-beta1 (TGF-beta1) mRNA in cultured rat mesangial cells. METHODS: (1) Mesangial cells were treated with medium containing different concentrations of ALD (10(-11), 10(-9), 10(-7) mol/L) and/or 10(-7) mol/L SPI for 48 h, while control cells were treated with vehicle only. The levels of FN in the supernatants were measured by ELISA method. The expressions of FN mRNA and TGF-beta1 mRNA were detected by semi-quantitative RT-PCR. (2) Mesangial cells were treated with 10(-9) mol/L ALD for 24, 48, 72 h. The levels of FN in the supernatants were measured by ELISA method. The expressions of FN mRNA and TGF-beta1 mRNA were detected by semi-quantitative RT-PCR. RESULTS: ELISA method showed that the level of FN in the supernatant of cultured rat mesangial cells stimulated with ALD increased significantly in a dose- and time-dependent manner. Also the expressions of FN mRNA and TGF-beta1 mRNA were increased significantly by ALD in a dose- and time-dependent manner by semi-quantitative RT-PCR. SPI inhibited the stimulating effect of ALD on synthesis of FN and expressions of FN mRNA and TGF-beta1 mRNA in cultured rat mesangial cells. CONCLUSION: ALD up-regulates the protein synthesis and mRNA expression of FN, and up-regulates the mRNA expression of TGF-beta1 in cultured rat mesangial cells. SPI inhibits the effect of ALD, and thus implication in the treatment of Glomerulosclerosis.     

Embryonic fibronectin isoforms are synthesized in crescents in experimental autoimmune glomerulonephritis.

Crescents are a severe and stereotyped glomerular response to injury that occur in several forms of glomerulonephritis that progress to renal failure. The key pathogenetic step that leads to glomerular scarring in unknown, but fibronectin (FN), the clotting system, macrophages, and proliferating parietal epithelial cells are known to participate. This study was designed to determine whether FN is synthesized locally, and in what molecular isoform, and whether cytokines known to promote FN synthesis are present in the crescent. Rats immunized with bovine glomerular basement membrane develop cellular crescents by 14 days and fibrous crescents and glomerulosclerosis by 35 days. In situ hybridization was performed with oligonucleotides specific for sequences common to all FN isoforms (total FN) or sequences specific for the alternatively spliced segments (EIIIA, EIIIB, and V). Throughout the time period (14, 21, and 35 days) all crescents and glomerular tufts contained cells with strong ISH signals for total and V+ mRNA, with the strongest signals present in large cellular crescents at day 21. In contrast, EIIIA+ and EIIIB+ mRNAs showed maximal abundance within sclerosing crescents at 35 days. Protein deposition of EIIIA+, EIIIB+, and V+ FN isoforms was confirmed by immunofluorescence with segment-specific FN antibodies. Transforming growth factor-beta and interleukin-1 beta, both known to promote FN synthesis, were found in cellular crescents (days 14 and 21) and were still present, but greatly diminished, in the sclerotic phase (day 35). In summary, EIIIA-, EIIIB-, and V+ FN mRNA plasma isoforms predominate in cellular crescents, whereas in the fibrosing stage, mainly the oncofetal EIIIA+, EIIIB+, and V+ isoforms are synthesized and accumulate.     

Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-beta activity.

The arterial response to injury is characterized by a short period of increased proliferation and migration of vascular smooth muscle cells, followed by an extended period of extracellular matrix accumulation in the intima. Transforming growth factor-beta (TGF-beta) has been implicated as a causative factor in the formation of extracellular matrix in this process, which leads to progressive thickening of the intima, known as intimal hyperplasia. In vitro analysis of vascular smooth muscle cells harvested from normal rat aortas and from aortas injured 14 days earlier showed that both types of cells attached equally well to culture dishes but that the initial spreading of the cells was increased in cells derived from injured vessels. Cells from the injured arteries produced more fibronectin and proteoglycans into the culture medium than the cells from normal arteries and contained more TGF-beta 1 mRNA. TGF-beta 1 increased proteoglycan synthesis by normal smooth muscle cells, and the presence of a neutralizing anti-TGF-beta 1 antibody reduced proteoglycan synthesis by the cells from injured arteries in culture. Fibronectin synthesis was not altered by these treatments. These results indicate that the accumulation of extracellular matrix components in neointimal lesions is at least partially caused by autocrine TGF-beta activity in vascular smooth muscle cells.     

Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity.     

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.     

Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta

Endothelial cells, including human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC), and bovine capillary endothelial cells (BCEC) in culture synthesize basic fibroblast growth factor (bFGF) and transforming growth factor type beta (TGF-beta). Basic FGF was cell-associated and synthesis was demonstrated by (i) the presence of bFGF mRNA species, (ii) binding to heparin-Sepharose and elution at 1.5 M NaCl, (iii) cross-reactivity with anti-bFGF antibodies when analyzed by electrophoretic blotting, and (iv) biological activity. Basic FGF was found in cell lysates at 2.3 ng/10(6) cells in HUVEC, 2.0 ng/10(6) cells in BCEC, and 13 ng/10(6) cells in BAEC. TGF-beta was secreted into media, and synthesis was demonstrated by (i) presence of TGF-beta mRNA species, (ii) cross-reactivity with anti-TGF-beta antibodies when analyzed by immunoprecipitation, (iii) competitive binding with authentic human platelet-derived TGF-beta that was blocked by TGF-beta specific blocking antibodies, and (iv) inhibition of [3H]TdR incorporation in CCl-64 cells. TGF-beta was secreted in an inactive form and required acid activation for detection. HUVEC synthesized 2.0 ng TGF-beta/10(6) cells per 12 hr; BCEC, 3.5 ng; and BAEC, 3.5 ng. HUVEC proliferation was not affected by treatment with exogenous TGF-beta, while BCEC proliferation was decreased by treatment with TGF-beta. Vascular endothelium is thus a source for these two potent multifunctional regulatory molecules, both of which may affect the growth of endothelium and neighboring fibroblasts, smooth muscle cells and white blood cells. The activation or release of these factors by endothelium may be a precipitating event in important cellular processes such as wound healing, organogenesis, and angiogenesis.     

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [(35)S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.     

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [³⁵S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.     

实验性肺纤维化大鼠组织整合素α5β1和转化生长因子—β的表达

目的 研究纤维连接蛋白受体整合蛋白受体整合素α５β１在大鼠肺纤维化中的作用。方法 用免疫组化方法观察实验性大鼠肺纤维化纤维连接蛋白及其受体整合素α５β１和转化生长因子－β表达的动态变化：用Ｎｏｒｔｈｅｍ印迹杂交和免疫细胞化学方法观察ＴＧＦ－β对体外培养的大鼠肺成纤维细胞整合素α５β１ ｍＲＮＡ和 白表达的影响。结果 （１）实验组１￣３天,病灶内上皮细胞和骨皮细胞整合素α５β１表达明显增强,炎细胞及     

Changes in the pattern of fibronectin mRNA alternative splicing in acute experimental mesangioproliferative nephritis

Fibronectins (FN) regulate cell migration, proliferation, and matrix formation during tissue injury. In humans, up to 20 different FN isoforms are generated by alternative splicing in three regions called EIIIA, EIIIB, and V, which have been implicated in the process undergoing wound healing and embryonic development. Specifically, EIIIA- and EIIIB-containing isoforms have been implicated in the regulation of cell proliferation and migration, whereas FN isoforms containing the full-length V region (named V120) are ligands to the VLA-4 integrin. To study the changes in the expression of FN isoforms in the anti-Thy-1 nephritis, an acute and self-resolutive model of mesangioproliferative nephritis, we analyzed the FN splicing patterns by means of ribonuclease protection assays. At Day 7 after anti-Thy-1 monoclonal injection, time of the maximal matrix expansion and glomerular hypercellularity, EIIIA+, EIIIB-, and V120 FN mRNA isoforms were increased. In accordance with the mRNA studies, FN proteins, including the EIIIA and V120 regions, increased in the mesangium of nephritic rats, as assayed by immunohistochemistry. Coinciding with the EIIIA and V120 isoforms up-regulation, there was an increase in mesangial cell proliferation and in the number of VLA-4+ infiltrating cells. At Day 14, in parallel with a remission of the above-mentioned changes, there was a decline in the mRNA and protein FN isoforms increased in the previous phase. The marked and reversible changes in the pattern of FN isoforms and their temporal association with other indicators of glomerular injury suggest that certain FN isotypes are important and coordinated components of the mechanisms attempting to reverse glomerular damage.     

Growth promoting substrates for human dermal fibroblasts provided by artificial extracellular matrices composed of collagen I and sulfated glycosaminoglycans

The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8-48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a "proliferative phenotype" of dFb found in the early stages of cutaneous wound healing.