Fn Staining Research Articles

Beneficial effect of all-trans retinoic acid (ATRA) on glomerulosclerosis rats via the down-regulation of the expression of α-smooth muscle actin: A comparative study between ATRA and benazepril

Although ATRA is a potent renoprotective agent, relatively little is known regarding the mechanisms of its action. The present study was designed to further elucidate the mechanisms of ATRA's action to GS rats and compare that with the beneficial effect of benazepril. Male SD rats weighting 160 to 200 g were used in this study. GS was induced by unilateral nephrectomy and intravenous injection of adriamycin (6 mg/kg). They were divided randomly 20 ones per group into GS group, GS treated with ATRA (20 mg/kg/day) group, and GS treated with benazepril (10 mg/kg/day) group. The other 20 ones were taken as sham-operation group, injected normal saline into caudal vein. 12 weeks later, all rats were subjected to sacrifice. As expected, the GS group exhibited significant lower serum TP and Alb, and higher BUN, Cr and proteinuria than those of the sham group. Administration of ATRA or benazepril did ameliorate these above disorders of biochemical parameters in GS rats. Extensive renal damage was observed in the GS group, such as mononuclear infiltration, mesangial proliferation, focal segment glomerular sclerosis, and tubulointerstitial fibrosis. The pathological changes in both ATRA and benazepril group were alleviated remarkably. Semiquantitative GSI was used to evaluate the degree of GS in all groups. GSI was significantly higher in the GS group than in sham group. GSI decreased from 21.9 ± 6.7 in the GS group to 6.9 ± 2.8 in the ATRA group and 7.0 ± 2.7 in benazepril group respectively. However, no significant difference in GSI between rats treated with ATRA and rats treated with benazepril was found. RT-PCR analysis revealed the renal expression of α-SMA mRNA was induced substantially in GS group as compared to sham group, which could be offset completely by ATRA or benazepril administration. However, expression level of α-SMA mRNA in GS rats treated with ATRA was identical to that in GS rats treated with benazepril. We also examined immunohistochemical staining for renal α-SMA, TGF-β1, Col IV, and FN in this model. Weak staining was observed in some glomerulus, mesangial cells, and tubular interstitium of sham rats. Staining was markedly enhanced in the majority of glomerulus, mesangial cells, and tubular interstitium of untreated GS rats. Compared with untreated GS animals, intensity and extent of staining for renal α-SMA, TGF-β1, Col IV, and FN were markedly reduced in glomerulus, mesangial cells, and tubular interstitium of GS rats treated with either ATRA or benazepril. However, no significant differences existed between ATRA and benazepril with respect to the glomerular and tubulointerstitial staining scores. Interestingly, our data documented some differences of therapeutic capacities between ATRA and benazepril. In comparison with benazepril, ATRA exerted no improvement in hypoproteinemia, but more significant decrease in serum Cr level in GS rats. The reasons leading to these variations are unclear. Whatever they are, the properties of down-regulate inflammatory/proliferative programs may make ATRA an attractive potential candidate for future therapeutic use in kidney disease.

Predicting Fibrosis Worsening in Obese Patients With NASH Through Parenchymal Fibronectin, HOMA-IR, and Hypertension

Few published studies have examined the results obtained from repeat liver biopsies in obese patients with nonalcoholic fatty liver disease (NAFLD). The progressive form of this disease may be largely limited to a subgroup of NAFLD patients with nonalcoholic steatohepatitis (NASH). The presence of intralobular fibronectin (Fn) and other variables was investigated in relation to subsequent fibrosis progression. In this prospective study, 271 obese patients admitted to the hospital with NAFLD and abnormal liver enzymes were scheduled to undergo a repeat liver biopsy at least 5 years after the initial biopsy. After excluding cirrhotic patients, basal biopsy specimens obtained from patients who underwent a second liver biopsy were stained with antibodies against Fn. The progression of fibrosis in the follow-up sample was correlated with the amount of Fn and other clinicopathological variables. We obtained a second liver biopsy from 149 patients after a median time of 6.4 years. Of these, 132 showed suitable Fn staining for semi-quantitative assessments. In all, 44 out of 83 patients (53%) with basal NASH showed fibrosis progression by at least one stage in the second liver biopsy. The amount of Fn (odds ratio=14.1; P<0.001), a diagnosis of hypertension (odds ratio=4.8; P=0.028), and homeostasis model assessment parameter of insulin resistance (HOMA-IR) scores (>8, odds ratio=1.9; P=0.004) were independent predictive factors of worsening fibrosis. A semi-quantitative assessment of the amount of parenchymal Fn present at an early stage in obese patients with NASH is valuable for predicting the progression of fibrosis. Similarly, lobular Fn deposition may be a sensitive and early indicator of active fibrogenetic processes in the liver. Hypertension and higher HOMA-IR scores are other clinical independent risk factors that predict the progression of fibrosis.

Expression of fibronectin and HIF-1α in renal cell carcinomas: relationship to von Hippel-Lindau gene inactivation

The von Hippel-Lindau ( VHL) tumor suppressor gene (TSG) at 3p25 is mutated in approximately 50% of conventional (clear cell) renal cell carcinomas (cRCC). VHL normally regulates the ubiquitin-mediated proteolysis of hypoxia-inducible factor 1α (HIF-1α), and VHL inactivation results in increased cellular HIF-1α expression. VHL protein (pVHL) also interacts with fibronectin (Fn) and VHL inactivation results in defective Fn extracellular matrix assembly. The present study investigated the immunohistochemical (IHC) staining for Fn and HIF-1α in 11 cRCC and the relationship of the staining to VHL inactivation by gene deletion, mutation, or hypermethylation. Evidence for VHL inactivation by 3p deletions and VHL mutations were found in six tumors. Fn-positive IHC staining of tumor cells and negative to weak staining of extracellular stroma was found in five cases having exon 1 or exon 2 mutations. In contrast, Fn staining was absent in tumor cells and positive in the stroma of five tumors without VHL inactivation and in one tumor with a C-terminal exon 3 mutation. HIF-1α tumor cell staining was present in the cRCC with VHL inactivation but was also present in two tumors having 3p deletions but neither mutation nor hypermethylation of VHL. These two cRCC showed a tumor cell–negative and stroma-positive pattern of Fn staining. The findings indicate that VHL inactivation plays a role in the development of some cRCC by altering Fn cell–stroma relationships. They also suggest that some C-terminal mutations may not interfere with Fn assembly and that a 3p TSG in addition to VHL influences HIF-1α degradation.

Study on the specificity of fibronectin for post-mortem diagnosis of early myocardial infarction.

In order to investigate the specificity of fibronectin (Fn) in the post-mortem diagnosis of myocardial infarction, the changes of Fn staining in normal, infarcted and other non-infarcted myocardial injuries resulting from myocarditis, mechanical asphyxia, electrocution, hemorrhagic shock, cardiac contusion and organophosphate poisoning were studied with an immunohistochemistry and image analysis system. The results showed that positive Fn staining could only be observed in groups of myocardial infarction and myocarditis, but could not be found in groups of mechanical asphyxia, electrocution, hemorrhagic shock, cardiac contusion, and organophosphate poisoning. Our findings indicate that positive staining of Fn in cardiomyocytes could be affected only by myocarditis, so it is quite specific for the diagnosis of myocardial infarction.

Immunocytochemical characterisation of rabbit and mouse embryonic fibroblasts.

By using indirect immunofluorescence we demonstrated the localisation of extracellular matrix (ECM) proteins (laminin--LAM, collagen IV--COL IV, fibronectin--FN) and the basic fibroblast growth factor (bFGF) in rabbit and mouse primary embryonic fibroblasts (PEF). Proliferating mitotically arrested PEF (by mitomycin C) were compared in both species. The stability of protein expression was ascertained during the first five successive passages. In addition, STO cells (i.e. permanent line of irradiated mouse fibroblasts) were similarly analysed. Rabbit PEF showed very high extracellular staining for FN and a negligible cytoplasmic positivity for LAM and COL IV. A totally reversed staining pattern for ECM proteins was found in mouse PEF. A dense cytoplasmic granulation (concentrated around the nucleus) was revealed for LAM and COL IV and almost no reaction for FN. The staining patterns were very stable at the culture conditions we applied. They were maintained during the first five successive passages in proliferating as well as non-proliferating mouse and rabbit PEF and were independent of cell concentration (individually dispersed cells versus cells in a confluent layer). STO cells showed the same staining for ECM proteins as the mouse PEF, thus confirming their origin from the same animal species. Light granular staining for bFGF was found in the cytoplasm of proliferating and mitotically arrested rabbit and mouse PEF and STO cells. The differences in expression of ECM proteins between the rabbit and mouse PEF, as well as the synthesis of bFGF, should be taken into consideration when these cells are used in vitro as a feeder layer for various cells (e.g. embryonic stem cells).

Analysis of fibronectin on human sperm.

The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = -0.68), motility (r = -0.70), and morphology (r = -0.63). This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm is primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Analysis of Fibronectin on Human Sperm

Purpose The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. Materials and Methods Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. Results FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = −0.68), motility (r = −0.70), and morphology (r = −0.63). Conclusions This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm in primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Alterations in the Localization of F-Actin, Fibronectin, and Thrombospondin Occur Prior to Neoplastic Transformation in Rat Tracheal Epithelial Cells

Structural glycoproteins and cytoskeletal proteins play a major role in the regulation of cellular organization and function. Changes in the structure and function of these proteins are involved in the cascade of events which lead to neoplastic transformation. We evaluated RNA levels, protein localization, and organization of selected proteins in an in vitro model system of respiratory carcinogenesis to examine alterations in cell architecture. Localization of fibronectin (Fn), thrombospondin (Tsp), and F-actin was examined in (1) primary rat tracheal epithelial (RTE) cells; (2) spontaneously immortalized nonneoplastic cells (SPOC-1); and (3) neoplastic cells (EGV5T) derived from tumors arising following transplantation of an N -methyl- N′-nitro- N-nitrosoguanidine-transformed RTE cell line into nude mice. Proteins were stained with fluorescein-labeled antibodies or phalloidin compound and analysis was performed with a confocal laser scanning microscope. Primary RTE cells display organized F-actin stress fibers, perinuclear Fn and Tsp, and pericellular Fn in fibrillar arrays. In larger colonies, Tsp occurs between cells and occasionally in fibrillar arrays. SPOC-1 cells, unlike primary RTE cells and neoplastic EGV5T cells, seldom form junctions and exhibit few cell surface extensions. F-actin stress fibers are reduced in these immortalized cells. F-actin in SPOC-1 cells occurs in the perinuclear region, scattered diffusely throughout the cell and in punctate adhesions. Fn and Tsp are localized to the perinuclear region with Fn staining more intensely. EGV5T neoplastic cells also display a dramatic loss of stress fibers and F-actin is concentrated mainly near the cell periphery. Perinuclear staining of Fn and Tsp occurs in some cells within the colony. Levels of Tsp RNA and Fn RNA and protein are significantly reduced in both cell lines compared to primary RTE cells. We conclude that structural protein disruptions are early events in the transformation of these respiratory epithelial cells.

Cloning and Characterization of Human Gingival and Periodontal Ligament Fibroblasts

Fibroblast heterogeneity in human periodontal tissues was characterized by cloning and immuno-histochemical techniques. Cell suspensions from primary cultures gingival (GF) and PDL fibroblasts (PF) were cloned. The relative intensity of double-labeled immunofluorescence, using specific antibodies to the extracellular matrix (ECM) molecules collagen type I (CI), type III (CIII), and fibronectin (Fn), was measured by photometry. Most clones derived from either GF or PF showed positive intracellular staining for both CI and Fn, and CIII and Fn. However, there were variations in fluorescence intensity for CI and Fn, ranging from relatively weak to strongly positive. The fluorescence for CI and CIII was relatively weak in most isolated GF clones in contrast to their PF clones. These observations coupled with studies of growth and cellular morphology in individual clones suggest that: 1) GF and PF contain functionally heterogeneous subpopulations; and 2) the synthesis and expression of extracellular matrix molecules of GF may be essentially different from that of PF.

Presence and distribution of fibronectin on the surface of implanted intraocular lenses in rabbits

An immunohistochemical study was performed to observe cellular proliferation on the surface of implanted intraocular lenses (IOLs) in rabbit eyes. Rabbits were killed at intervals of 3 days and 1, 2, and 4 weeks after the operation. The IOLs were removed and examined by an immunoperoxidase staining method using antifibronectin (anti-FN) antibodies. The FN immunoreactivity was detected in macrophages and giant cells attached to the IOL surface. Prominent staining was observed in these cells 1 week after the operation, and staining for FN was less intense in the specimens obtained 2 and 4 weeks after implantation. These findings suggest that FN is produced by macrophages and giant cells on the IOL surface and may play an important role in cellular adhesion and motility. FN immunoreactivity decreased with time, which might be related to cellular activity.

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by (1) testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and (2) to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 μm in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In constrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower M r proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.

Thrombin increases expression of fibronectin antigen on the platelet surface.

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

The detection, immunofluorescent localization, and thrombin induced release of human platelet‐associated fibronectin antigen

Platelets are cells which develop adhesive properties following stimulation. Since fibronectin (fn) mediates adhesive properties of several cells, we sought evidence for platelet associated fn. Lysates of suspensions of washed human platelets containing less than or equal to 50 ng soluble fn/10(9) cells contained 2.85 micrograms fn antigen per 10(9) cells. The platelet fn antigen competition curve showed a similar slope to the curve for purified plasma fn suggesting antigenic identity. Immunofluorescent staining for fn was minimal in intact cells suggesting that the majority of fn antigen is intracellular. In permeable platelets, fluorescent staining for fn was seen in a punctate distribution suggesting a granule localization. Stimulation of platelet secretion by thrombin released platelet fn antigen. Suramin, a drug which inhibits platelet secretion, inhibited fn release. The apparent secretion of platelet fn, taken with the immunofluorescent data, support the localization of a portion of platelet fn antigen in a storage granule.