ABSTRACT
BACKGROUND AND AIM: Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats. METHODS: 8 weeks after 6-OHDA injection animals were sacrificed. Inflammatory infiltrates, collagen deposition and remodelling of intestinal epithelial barrier and tunica muscularis in the colonic wall were assessed by histochemistry, immunohistochemistry, immunofluorescence and western blot analysis. RESULTS: 6-OHDA rats displayed significant alterations of colonic tissues as compared with controls. Signs of mild inflammation (eosinophil infiltration) and a transmural deposition of collagen fibres were observed. Superficial colonic layers were characterized by severe morphological alterations. In particular, lining epithelial cells displayed a reduced claudin-1 and transmembrane 16A/Anoctamin 1 (TMEM16A/ANO1) expression; goblet cells increased their mucin expression; colonic crypts were characterized by an increase in proliferating epithelial cells; the density of S100-positive glial cells and vimentin-positive fibroblast-like cells was increased as well. Several changes were found in the tunica muscularis: downregulation of α-smooth muscle actin/desmin expression and increased proliferation of smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia; reduction of interstitial cells of Cajal (ICCs) density. CONCLUSIONS: A pathological remodelling occurs in the colon of 6-OHDA rats. The main changes include: enhanced fibrotic deposition; alterations of the epithelial barrier; activation of mucosal defense; reduction of ICCs. These results indicate that central nigrostriatal denervation is associated with histological changes in the large bowel at mucosal, submucosal and muscular level. These alterations might represent morphological correlates of digestive symptoms in PD.
Subject(s)
Colon/pathology , Dopaminergic Neurons/pathology , Animals , Anoctamin-1 , Colon/metabolism , Dopamine/metabolism , Fibrosis , Gastrointestinal Diseases/metabolism , Gastrointestinal Motility , Male , Oxidopamine , Parkinson Disease/metabolism , Parkinson Disease/pathology , Rats , Rats, Sprague-Dawley , Substantia NigraABSTRACT
Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.
Subject(s)
Heart Neoplasms/pathology , Laser Capture Microdissection/methods , Microscopy, Confocal/methods , Myocardium/pathology , Myxoma/pathology , Actins/biosynthesis , Actins/genetics , Adult , Aged , Aged, 80 and over , Calbindin 2/biosynthesis , Calbindin 2/genetics , Cell Differentiation , Female , Gene Expression Regulation, Neoplastic , Heart Neoplasms/genetics , Heart Neoplasms/surgery , Humans , Immunohistochemistry , Male , Middle Aged , Myocardium/metabolism , Myxoma/genetics , Myxoma/surgery , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Platelet Endothelial Cell Adhesion Molecule-1/genetics , RNA, Neoplasm/genetics , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND AND AIMS: Intestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. METHODS: Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS: Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. CONCLUSIONS: A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.
Subject(s)
Colitis, Ulcerative/pathology , Colon/pathology , Vascular Remodeling , Acute Disease , Adult , Aged , Biomarkers/metabolism , Blotting, Western , Case-Control Studies , Colon/blood supply , Colon/metabolism , Disease Progression , Female , Fibrosis , Humans , Male , Middle Aged , Retrospective Studies , Single-Blind MethodABSTRACT
Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.
Subject(s)
Colitis/metabolism , Collagen/metabolism , Colon/metabolism , Coloring Agents , Immunohistochemistry , Animals , Colitis/pathology , Immunohistochemistry/methods , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , RatsABSTRACT
Gap junctions (GJ) mediate electrical coupling between cardiac myocytes, allowing the spreading of the electrical wave responsible for synchronized contraction. GJ function can be regulated by modulation of connexon densities on membranes, connexin (Cx) phosphorylation, trafficking and degradation. Recent studies have shown that adenosine (A) involves Cx43 turnover in A1 receptor-dependent manner, and dipyridamole increases GJ coupling and amount of Cx43 in endothelial cells. As the abnormalities in GJ organization and regulation have been described in diseased myocardium, the aim of the present study was to assess the regional expression of molecules involved in GJ regulation in a model of left ventricular dysfunction (LVD). For this purpose the distribution and quantitative expression of Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, RhoA and A receptors, were investigated in experimental models of right ventricular-pacing induced LVD, undergoing concomitant dipyridamole therapy or placebo, and compared with those obtained in the myocardium from sham-operated minipigs. Results demonstrate that an altered pattern of factors involved in Cx43-made GJ regulation is present in myocardium of a dysfunctioning left ventricle. Furthermore, dipyridamole treatment, which shows a mild protective role on left ventricular function, seems to act through modulating the expression and activation of these factors as confirmed by in vitro experiments on cardiomyoblastic cell line H9c2 cells.
Subject(s)
Connexin 43/metabolism , Dipyridamole/therapeutic use , Gap Junctions/drug effects , Heart Ventricles/drug effects , Ventricular Dysfunction, Left/drug therapy , Animals , Cell Line , Connexin 43/genetics , Dipyridamole/administration & dosage , Disease Models, Animal , Electrocardiography , Gap Junctions/metabolism , Gap Junctions/pathology , Heart Ventricles/metabolism , Heart Ventricles/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Phosphorylation , Rats , Signal Transduction , Swine , Swine, Miniature , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/pathologyABSTRACT
Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.
Subject(s)
Colitis/pathology , Myocytes, Smooth Muscle/pathology , Animals , Colon/pathology , Inflammation/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD. METHODS AND FINDINGS: By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs. The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors. CONCLUSION: This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.
Subject(s)
Colon/metabolism , Diverticulum, Colon/metabolism , Muscle, Smooth/metabolism , Adult , Aged , Aged, 80 and over , Colon/pathology , Connexin 26 , Connexin 43/genetics , Connexin 43/metabolism , Connexins/genetics , Connexins/metabolism , Diverticulum, Colon/genetics , Diverticulum, Colon/pathology , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Male , Middle Aged , Muscle, Smooth/pathology , Protein Kinase C/genetics , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
Gastrointestinal neuromuscular diseases (GINMDs) comprise a heterogenous group of chronic conditions associated with impaired gut motility. These gastrointestinal (GI) disorders, differing for etiopathogenic mechanisms, pathologic lesions, and region of gut involvement, represent a relevant matter for public health, because they are very common, can be disabling, and determine major social and economic burdens. GINMDs are presumed or proven to arise as a result of a dysfunctioning GI neuromuscular apparatus, which includes myenteric ganglia (neurons and glial cells), interstitial cells of Cajal and smooth muscle cells. Despite the presence of symptoms related to gut dysmotility in the clinical phenotype of these patients, in the diagnostic setting scarce attention is usually paid to the morphologic pattern of the GI neuromuscular apparatus. It is also objectively difficult to collect full-thickness gut tissue samples from patients with GINMDs, because their disease, which can be only functional in nature, may not justify invasive diagnostic procedures as a first-line approach. As a consequence, whenever available, bioptic gut specimens, retrieved from these patients, must be regarded as a unique chance for obtaining relevant diagnostic information. On the basis of these arguments, there is an urgent need of standardized and validated histopathologic methods, aiming at overcoming the discrepancies affecting current approaches, which usually lead to conflicting definitions of normality and hamper the identification of disease-specific pathologic patterns. This review article intends to address current methodological and ontological issues in the histopathologic diagnosis of GINMDs, to foster the debate on how to discriminate normal morphology from abnormalities.
Subject(s)
Gastrointestinal Diseases/etiology , Gastrointestinal Diseases/pathology , Neuromuscular Diseases/etiology , Neuromuscular Diseases/pathology , Chronic Disease , Enteric Nervous System/physiopathology , Gastrointestinal Diseases/physiopathology , Gastrointestinal Motility/physiology , Humans , Neuromuscular Diseases/physiopathologyABSTRACT
Ulcerative colitis (UC) is an inflammatory bowel disease with alterations of colonic motility, which influence clinical symptoms. Although morpho-functional abnormalities in the enteric nervous system have been suggested, in UC patients scarce attention has been paid to possible changes in the cells that control colonic motility, including myenteric neurons, glial cells and interstitial cells of Cajal (ICC). This study evaluated the neural-glial components of myenteric ganglia and ICC in the colonic neuromuscular compartment of UC patients by quantitative immunohistochemical analysis. Full-thickness archival samples of the left colon were collected from 10 patients with UC (5 males, 5 females; age range 45-62 years) who underwent elective bowel resection. The colonic neuromuscular compartment was evaluated immunohistochemically in paraffin cross-sections. The distribution and number of neurons, glial cells and ICC were assessed by anti-HuC/D, -S100ß and -c-Kit antibodies, respectively. Data were compared with findings on archival samples of normal left colon from 10 sex- and age-matched control patients, who underwent surgery for uncomplicated colon cancer. Compared to controls, patients with UC showed: (i) reduced density of myenteric HuC/D(+) neurons and S100ß(+) glial cells, with a loss over 61% and 38%, respectively, and increased glial cell/neuron ratio; (ii) ICC decrease in the whole neuromuscular compartment. The quantitative variations of myenteric neuro-glial cells and ICC indicate considerable alterations of the colonic neuromuscular compartment in the setting of mucosal inflammation associated with UC, and provide a morphological basis for better understanding the motor abnormalities often observed in UC patients.
Subject(s)
Colitis, Ulcerative/pathology , Ganglia/pathology , Interstitial Cells of Cajal/pathology , Myenteric Plexus/pathology , Colitis, Ulcerative/metabolism , Colon/metabolism , Colon/pathology , ELAV Proteins/metabolism , ELAV-Like Protein 3 , Female , Ganglia/metabolism , Humans , Interstitial Cells of Cajal/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Male , Middle Aged , Nerve Growth Factors/metabolism , Neuroglia/metabolism , Neuroglia/pathology , Neurons , S100 Calcium Binding Protein beta Subunit , S100 Proteins/metabolismABSTRACT
The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon (n = 10, age-range: 50-72 years) were examined. HuC/D and S100beta antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100beta-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens.
Subject(s)
Colon/cytology , Ganglia/cytology , Myenteric Plexus/cytology , Aged , Cell Count , Colon/metabolism , ELAV Proteins/metabolism , Female , Ganglia/metabolism , Humans , Immunohistochemistry , Male , Middle Aged , Myenteric Plexus/metabolism , Neurofilament Proteins/metabolism , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Neurons/metabolism , Regression AnalysisABSTRACT
BACKGROUND: Osteogenic growth peptide (OGP) is an endogenous tetradecapeptide present in micromolar concentrations in mammalian serum; its carboxy-terminal pentapeptide, OGP(10-14), represents its physiologically active fragment. OGP(10-14) induces proliferation and differentiation in fibroblast and osteoblast cell lines, and it enhances hematopoiesis in vitro and in vivo. The signaling pathways triggered by OGP(10-14) are not yet fully known. In the present report, we evaluated the effect of OGP(10-14) on differentiation of a cancer megakaryoblast cell line and its involvement on RhoA and Src family kinases signaling pathway. MATERIAL/METHODS: Cell proliferation of the Mo-7e line was evaluated using the MTT test. Mo-7e differentiation was evaluated by microscopic observation of cell morphology and by expression of the factor VIII-related antigen. Involvement of RhoA and Src kinases on signaling pathways triggered by OGP(10-14) was analyzed using RhoA and Src family kinase (SFK) inhibitors (C3 and PP2) and an immunoperoxidase technique. RESULTS: OGP(10-14) induces expression of the factor VIII-related antigen, morphologic changes indicative of megakaryocytic differentiation, and a down-regulation of the Fyn Src kinase. These OGP(10-14) effects were prevented by C3 and enhanced by PP2. CONCLUSIONS: The anti-proliferative and pro-differentiating activities of OGP(10-14) on thrombopoietin (TPO)-primed Mo-7e cells are mediated by RhoA and Src kinase pathways as demonstrated by the use of C3 and PP2.
Subject(s)
Histones/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , rhoA GTP-Binding Protein/metabolism , src-Family Kinases/metabolism , ADP Ribose Transferases/pharmacology , Botulinum Toxins/pharmacology , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Humans , Immunohistochemistry , Proto-Oncogene Proteins c-fyn/metabolism , Pyrimidines/pharmacology , von Willebrand Factor/metabolismABSTRACT
Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].
Subject(s)
Gelatin , Lactic Acid , Mesenchymal Stem Cells/cytology , Osteogenesis , Polymers , Tissue Engineering/methods , Cell Differentiation , Cell Proliferation , Humans , Polyesters , Stromal Cells/cytologyABSTRACT
Tissue engineering scaffolds should be able to reproduce optimal microenvironments in order to support cell attachment, three-dimensional growth, migration and, regarding fibroblasts, must also promote extracellular matrix production. Various bioactive molecules are employed in the preparation of spongy scaffolds to obtain biomimetic matrices by either surface-coating or introducing them into the bulk composition of the biomaterial. The biomimetic properties of a spongy matrix composed of PVA combined with the natural component gelatine were evaluated by culturing human gingival fibroblasts on the scaffold. Cell adhesion, morphology and distribution within the scaffold were assessed by histology and electron microscopy; viability and metabolic activity as well as extracellular matrix production were analyzed by MTT assay, cytochemistry and immunocytochemistry. Fibroblasts interacted positively with PVA/gelatine. They adhered to the PVA/gelatine matrix in which they had good spreading activity and active metabolism; fibroblasts were also able to produce extracellular matrix molecules (type I collagen, fibronectin and laminin) compared to bi-dimensionally grown cells. The in situ creation of a biological matrix by human fibroblasts together with the ability to produce growth factor TGF-beta1 and the intracellular signal transduction molecule RhoA, suggests that this kind of PVA/gelatine sponge may represent a suitable support for in vitro extracellular matrix production and connective tissue regeneration.
Subject(s)
Extracellular Matrix/metabolism , Fibroblasts/physiology , Gelatin , Gingiva/cytology , Polyvinyl Alcohol , Tissue Engineering/methods , Cell Adhesion , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/ultrastructure , Humans , Immunohistochemistry , Microscopy, Electron/methods , Transforming Growth Factor beta1/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
The low density lipoprotein (LDL) receptor-associated protein (RAP) is an endoplasmic reticulum (ER)-resident molecular chaperone for several LDL receptor family members and it also binds to thyroglobulin (Tg), the thyroid hormone precursor. Disruption of the RAP gene in thyrocytes results in impaired Tg secretion. To gain further insights into the function of RAP in the thyroid, we investigated whether its expression in thyrocytes is regulated by thyroid-stimulating hormone (TSH), a feature common to all proteins involved in thyroid hormone secretion. We found by immunofluorescence that in FRTL-5 cells cultured in the presence of TSH, RAP is expressed intracellularly. The levels of expression increased after exposure to TSH, beginning at 48 hours, in a concentration-dependent manner as observed by immunofluorescence and Western blotting. Expression of RAP was also increased by TSH in primary cultures of human thyrocytes as observed by Western blotting. In hypothyroid mice with high serum TSH, RAP was markedly increased compared with euthyroid mice as observed by immunohistochemistry and Western blotting. Based on these findings, we concluded that RAP is expressed by thyrocytes in a TSH-dependent manner, both in cultured thyroid cells and in vivo.
Subject(s)
Epithelial Cells/metabolism , LDL-Receptor Related Protein-Associated Protein/metabolism , Thyroid Gland/cytology , Thyrotropin/pharmacology , Animals , Antithyroid Agents/pharmacology , Blotting, Western , COS Cells , Chlorocebus aethiops , Epithelial Cells/cytology , Fluorescent Antibody Technique , Humans , Hypothyroidism/chemically induced , Hypothyroidism/metabolism , Methimazole/pharmacology , Mice , Perchlorates/pharmacology , Rats , Sodium Compounds/pharmacology , Thyroid Gland/metabolism , Up-Regulation/drug effectsABSTRACT
The aim of this research was to characterize the immediate alterations induced by angioplasty and to compare the results of the application of two types of balloons. Ten porcine renal arteries were dilated with a compliant balloon, and ten with a non-compliant balloon. After angioplastic treatment arterial specimens were wax embedded for light microscopy. Sections were stained with the orcein-Van Gieson method, orcein, haematoxylin-eosin, and PAS. Image analysis was performed taking into consideration the following parameters: thickness of the entire wall, of the tunica media and of the inner elastic lamina. The major axes of the smooth muscle cells nuclei were also measured. The effects of the two types of balloon resulted in changes consisting in thinning of the entire arterial wall, reduction of the tunica media, distension of reticular fibers, presence of wide spaces between smooth muscle cells, stretching of smooth muscle cells, inner elastic lamina thickening. Both angioplasty devices used can modify the vascular wall. The identification of the tunica media structural damages might be useful in order to estimate the behavior of the vascular wall in the follow-up after angioplasty, because the entity of modifications could be predictive of restenosis that often takes place weeks or months after angioplasty.
Subject(s)
Angioplasty, Balloon , Renal Artery/pathology , Animals , Cell Nucleus/pathology , Histocytochemistry , Image Cytometry , Muscle, Smooth, Vascular/pathology , Renal Artery/surgery , Swine , Tunica Intima/pathology , Tunica Media/pathologyABSTRACT
BACKGROUND: The Rho/transforming growth factor-beta (TGF-beta) system plays a crucial role in the progression of renal damage due to stimulation of extracellular matrix molecule deposition. In fact, the in vitro TGF-beta-mediated production of fibronectin, one of the major TGF-beta-regulated extracellular components, has recently been correlated with Rho protein signalling molecules. Although a close relationship between increased renal tissue levels of TGF-beta1 and fibronectin has been reported in IgA nephropathy, no data are available on renal tissue expression of Rho proteins. METHODS: This study was designed to assess in IgA nephropathy patients the kidney tissue immunohistochemical expression of RhoA, TGF-beta1, and fibronectin, and the rate of immunoreactivity for each antigen by image analysis. RESULTS: An increase in RhoA, TGF-beta1, and fibronectin expression was detected in tubulointerstitium and in glomeruli of IgA nephropathy compared to normal kidneys; in particular, RhoA was found also in proximal tubules, unlike control kidneys and mainly at the cell boundary level, which is in keeping with its activated form. The image analysis confirmed that the kidney tissue levels of RhoA, TGF-beta1, and fibronectin were significantly enhanced in the patients. CONCLUSION: This study suggests that RhoA may represent a key molecule in the signalling transduction pathway of profibrotic signals in IgA nephropathy.
Subject(s)
Fibronectins/biosynthesis , Glomerulonephritis, IGA/genetics , Glomerulonephritis, IGA/physiopathology , Transforming Growth Factor beta/analysis , rhoA GTP-Binding Protein/biosynthesis , rhoA GTP-Binding Protein/physiology , Adolescent , Adult , Antigens/analysis , Biopsy , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Kidney/physiology , Male , Middle Aged , Signal Transduction , Transforming Growth Factor beta/physiology , Transforming Growth Factor beta1ABSTRACT
Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.
Subject(s)
Low Density Lipoprotein Receptor-Related Protein-2/physiology , Thyroid Gland/physiopathology , Animals , Heterozygote , Homozygote , Low Density Lipoprotein Receptor-Related Protein-2/deficiency , Mice , Mice, Knockout , Thyroglobulin/blood , Thyroglobulin/metabolism , Thyroid Gland/metabolism , Thyroid Hormones/blood , Thyroid Hormones/metabolism , Thyrotropin/blood , Thyroxine/bloodABSTRACT
Rho proteins, a subgroup of the Ras GTPase superfamily, control many cellular processes and morphogenetic events by acting as signaling molecules in the transduction pathways of various receptors. Among the "Rho-dependent" receptors are the extracellular matrix- and growth factor-binding sites; these are particularly involved in the modulation of renal development since they control the epithelial-mesenchymal interactions that drive kidney organogenesis. The present study has addressed the immunohistochemical localization of RhoA in developing and adult kidneys of rats and humans because: a) Rho proteins are known to have a morphogenetic role, b) data in the literature on expression of Rho GTPases during mammalian histogenesis and organogenesis are scarce, and c) their involvement in the transduction pathways of receptors is implicated in kidney development. In particular, RhoA peptide was found to be localized in the mesonephric duct and vesicles in both rats and humans; metanephric anlagen were mainly stained in ampullar-derived cells. Periglomerular tubules of fetal and adult kidneys as well as collecting ducts of adult kidneys showed intense staining. Therefore, the present study provides new information on the distribution patterns of RhoA during early stages of mammalian kidney development suggesting that this signaling molecule may take part in epithelial-mesenchymal induction processes that control kidney organogenesis. RhoA expression in adult structures may be linked with renewal of renal epithelial cells and the maintenance of their morphology and polarity.