Distribution Of Extracellular Matrix Components Research Articles

Delineation of the healthy rabbit heart by immunohistochemistry – A technical note

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining.

Preconditioning process for dermal tissue decellularization using electroporation with sonication.

Decellularization to produce bioscaffolds composed of the extracellular matrix (ECM) uses enzymatic, chemical and physical methods to remove antigens and cellular components from tissues. Effective decellularization methods depend on the characteristics of tissues, and in particular, tissues with dense, complex structure and abundant lipid content are difficult to completely decellularize. Our study enables future research on the development of methods and treatments for fabricating bioscaffolds via decellularization of complex and rigid skin tissues, which are not commonly considered for decellularization to date as their structural and functional characteristics could not be preserved after severe decellularization. In this study, decellularization of human dermal tissue was done by a combination of both chemical (0.05% trypsin-EDTA, 2% SDS and 1% Triton X-100) and physical methods (electroporation and sonication). After decellularization, the content of DNA remaining in the tissue was quantitatively confirmed, and the structural change of the tissue and the retention and distribution of ECM components were evaluated through histological and histochemical analysis, respectively. Conditions of the chemical pretreatment that increase the efficiency of physical stimulation as well as decellularization, and conditions for electroporation and sonication without the use of detergents, unlike the methods performed in previous studies, were established to enable the complete decellularization of the skin tissue. The combinatorial decellularization treatment formed micropores in the lipid bilayers of the skin tissues while removing all cell and cellular residues without affecting the ECM properties. Therefore, this procedure can be widely used to fabricate bioscaffolds by decellularizing biological tissues with dense and complex structures.

Delineation of the healthy rabbit liver by immunohistochemistry – A technical note

Liver diseases pose a big global health problem and liver failure may result from viral infection, overnutrition or tumors. Studying pathologic liver tissue demands for accurate and specific histological stainings and immunohistochemical labellings, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy liver stainings and labellings are required, to provide a baseline or reference for the pathological situation.Here, we used the liver tissue of a healthy rabbit and compared different histological key steps, such as paraffin embedding after formalin fixation versus cryopreservation; or an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or chromogenic with fluorescent detection system, respectively. Moreover, we provide images of serial sections, where we stained the same morphological structure with different markers, including collagen I, collagen III, fibronectin, α-SMA, elastin, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, ki67 for proliferating cells, and orcein (as negative control for pathological aberrations like Wilson disease). Differences between conditions were quantitatively assessed by measuring the colour intensity.Generally, we observed that cryosections exhibited a stronger signal intensity in immunohistochemically labelled sections than in paraffin sections; however, the strong staining got slurred, which sometimes hampered proper identification of morphological structures at higher magnifications. Moreover, there was a clear increase in signal intensity for paraffin sections when an AR step was performed compared to condition without AR. Results for mouse isotype staining as a negative control clearly supported those findings.Different stainings of the portal triad, the central vein and the bile ducts revealed a clear-cut distribution of extracellular matrix components, with prominent fibronectin and elastin around the lumen of the central vein as well as a patchy PAR-2 expression. As for the bile ducts, complete absence of α-SMA and PAR-2 was found at the margins, however, collagen I expression and elastin were positive and showed a strong signal. Like this, we provide useful and valuable reference images for researchers using the rabbit liver model. It may help to decide which of the immunohistochemical protocols are valuable to reach a certain aim and which protocols lead to the best visualization of the target structure.

Is There a Scientific Rationale for the Refixation of Delaminated Chondral Flaps in Femoroacetabular Impingement? A Laboratory Study.

Debonding of the acetabular cartilage is a characteristic type of hip damage found in cam-type femoroacetabular impingement (FAI), which remains a treatment challenge. In addition to resection, refixation of these flaps using fibrin sealants has been recently suggested. However, there is only limited evidence available that the proposed refixation method results in sufficient viable cartilage formation to ensure long-term flap grafting and restored tissue function. To determine the flap tissue characteristics that would justify refixation of delaminated chondral flaps with a fibrin sealant, we characterized (1) the extracellular matrix (ECM) of chondral flaps in terms of chondrocyte viability and distribution of ECM components and (2) the chondrogenic potential of resident cells to migrate into fibrin and produce a cartilaginous matrix. Ten acetabular chondral flaps and three non-delaminated control cartilage samples were resected during surgery. Chondrocyte viability was quantified using a live-dead assay. To assess the ECM, histological staining of glycosaminoglycans, collagen II, and collagen I allowed the qualitative study of their distribution. The ability of chondrocytes to migrate out of the ECM was tested by encapsulating minced flap cartilage in fibrin gels and semi-quantitatively assessing the projected area of the gel covered with migrating cells. The potential of chondrocytes to produce a cartilaginous matrix was studied with a pellet assay, a standard three-dimensional culture system to test chondrogenesis. Positive controls were pellets of knee chondrocytes of age-matched donors, which we found in a previous study to have a good capacity to produce cartilage matrix. Statistical significance of controlled quantitative assays was determined by the Student's t-test with Welch's correction. The proportion of viable chondrocytes in flaps was lower than in nondelaminated cartilage (50% ± 19% versus 76 ± 6%; p = 0.02). Histology showed a disrupted ECM in flaps compared with nondelaminated controls, with the presence of fibrillation, a loss of glycosaminoglycan at the delaminated edge, collagen II throughout the whole thickness of the flap, and some collagen I-positive area in two samples. The resident chondrocytes migrated out of this disrupted ECM in all tested samples. However in pellet culture, cells isolated from the flaps showed a qualitatively lower chondrogenic potential compared with positive controls, with a clearly inhomogeneous cell and matrix distribution and an overall smaller projected area (0.4 versus 0.7 mm; p = 0.038). Despite the presence of viable chondrocytes with migration potential, the cells resided in a structurally altered ECM and had limited capacity to deposit ECM, leading us to question their capacity to produce sufficient ECM within the fibrin sealant for stable long-term attachment of such flaps. The characterization of delaminated cartilage in cam FAI patients suggests that the refixation strategy might be adversely influenced by the low level of ECM produced by the residing cells.

Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules.

The bone marrow (BM) microenvironment contains many types of cells and molecules with roles in hematopoiesis, osteogenesis, angiogenesis and metabolism. The spatial distribution of the different bone and BM cell types remains elusive, owing to technical challenges associated with bone imaging. To map nonhematopoietic cells and structures in bone and BM, we performed multicolor 3D imaging of osteoblastic, vascular, perivascular, neuronal and marrow stromal cells, and extracellular-matrix proteins in whole mouse femurs. We analyzed potential interactions between cells and molecules on the basis of colocalization of markers. Our results shed light on the markers expressed by different osteolineage cell types; the heterogeneity of vascular and perivascular cells; the neural subtypes innervating marrow and bone; the diversity of stromal cells; and the distribution of extracellular-matrix components. Our complete imaging data set is available for download and can be used in research in bone biology, hematology, vascular biology, neuroscience and extracellular-matrix biology.

Prolyl hydroxylase in human corpora lutea during menstrual cycle and early pregnancy

Immunohistochemical staining for human prolyl hydroxylase revealed intense staining of the human corpora lutea (CL) parencyma during early pregnancy compared with those in the menstrual cycle. These results suggest that human prolyl hydroxylase might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

Method for the decellularization of intact rat liver

We have developed a method for the decellularization of whole rat livers by perfusion with increasing concentrations of detergents. This procedure resulted in an intact, decellularized organ with an intact liver capsule. These decellularized organs were analyzed by immunohistochemistry, and retained an appropriate distribution of extracellular matrix components. The laminin basement membranes of the liver vasculature also remain intact. These acellular vessel remnants were strong enough to be cannulated; providing a convenient means for the delivery of cells to areas deep within the decellularized organ. Cannulation of the extrahepatic vessel remnants allow for media to be circulated through the decellularized organ. These decellularized livers provide a natural matrix for research in the fields of bio-artificial livers and liver engineering.

Distribution of Extracellular Matrix Components in Normal and Degenerated Canine Tricuspid Valve Leaflets

The aim of the present study was to investigate the composition and distribution of various extracellular matrix (ECM) components in normal canine tricuspid valves (TVs) and in TVs affected by chronic valvular disease (CVD). The parietal (pTV) and septal (sTV) leaflets of the TVs from 27 dogs were investigated immunohistochemically for expression of collagen types I, III, IV and VI, elastin, laminin, fibronectin and heparan sulphate. Normal pTV consisted mainly of elastin and collagen VI in the atrialis, fibronectin in the thin spongiosa and mixed collagens in the fibrosa. The layered structure was less distinct in sTV, with numerous adipocytes and proteoglycans in the spongiosa and collagen III predominating in the fibrosa. The earliest stages of CVD affecting the pTV were recognized in the spongiosa and progression to advanced disease was characterized by nodular accumulation of proteoglycans within the free edge of the leaflet. These nodular lesions of the pTV contained more fibronectin, elastin and collagens I and VI than those affecting the sTV. These findings contrast with those reported in CVD affecting the mitral valve (MV) in which the early lesions affect the atrialis and advanced disease involves the entire leaflet. The pathogenesis of CVD in TV may involve initial alterations of the tricuspid annulus that lead to early lesions within the spongiosa, resulting in further shear stress and proteoglycan accumulation at the free edge of the pTV.

Collagen Type-I α1 Chain mRNA is Expressed in the Follicle Cells of the Medaka Ovary

Follicle rupture during ovulation is a well-regulated biological process of extracellular matrix degradation in the vertebrate ovary. Although proteolytic enzymes responsible for the rupture have recently been identified in the medaka, Oryzias latipes , the lack of knowledge about the ovarian expression and distribution of extracellular matrix components in lower vertebrates prevents the understanding of this process's molecular mechanism. To approach the problem, we cloned a cDNA coding for the medaka collagen type-I alpha1 chain and examined its mRNA expression in the fish ovary. The deduced amino acid sequence of the collagen type-I alpha1 chain was homologous to those of the proteins from other vertebrate species. The alpha1 chain mRNA was expressed in various tissues of the adult fish. In the ovary sections of mature female fish, this mRNA was detected in a line surrounding ovarian follicles of all sizes. A comparison with the distribution of gelatinase B mRNA in follicles that had just ovulated indicated that the collagen type-I alpha1 gene is expressed in the theca cells. The current results strongly suggest that collagen type I is synthesized by theca cells and is localized in the same cell layer of the follicles.

Airway wall remodeling in asthma: From the epithelial layer to the adventitia

Asthma is an episodic respiratory syndrome caused by several pathogenic processes. This recurrent syndrome is associated with an accelerated decline in lung function and increase in airway obstruction over time. The reduced lung function is a consequence of tissue restructuring of all the components of the airway wall: 1) epithelium metaplasia; 2) altered quantity, composition, and distribution of extracellular matrix components; 3) microvascular remodeling; and 4) increase of airway smooth muscle mass. How these structural changes affect lung functions is not entirely clear. Deeper understandings of the altered structure and related functional impairment are important for gaining insights into the mechanisms underlying asthma. This review describes the tissue remodeling observed in different compartments of the asthmatic airway wall, from the airway lumen to adventitia. The underlying mechanisms driving the remodeling processes are also briefly reviewed.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Modulation of extracellular matrix components by metalloproteinases and their tissue inhibitors during degeneration and regeneration of rat sural nerve

The success of peripheral nervous system regeneration has been associated with changes on the microenvironment, particularly on the extracellular matrix (ECM) components. In the present study we analyzed by indirect immunohistochemistry, electron microscopy and Western blotting, the distribution of ECM components, metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), during Wallerian degeneration (WD) and nerve regeneration (2nd, 7th and 21st days after injury) on crushed rat sural nerves. Our results showed that laminin α 3-chain and α 2-chain are over expressed during the early stages of degeneration and regeneration respectively, whereas type IV collagen expression increased progressively after crush. On the other hand, the expression of chondroitin sulfate was down regulated during the early stages of degeneration, returning progressively to normal values during nerve regeneration. The expression of MMP-3 was almost normal immediately after lesion, and then reduced progressively achieving the smallest expression at 21 days after crush; on the contrary, the expression of MMP-7 increased significantly immediately after crush (2nd day) returning to normal values afterwards. TIMP-1 and TIMP-2 were over expressed at the beginning of WD, returning progressively to normal values after that. These results indicate that the modifications of ECM components, which are favorable for nerve regeneration, are correlated with changes on the balance between MMPs and TIMPs.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Extracellular matrix: Forum introduction

The extracellular matrix (ECM) is now appreciated as a key regulator of cell and tissue behavior. Support for this notion has been derived from correlative studies demonstrating large changes in the composition and distribution of extracellular matrix components during tissue development, differentiation and in response to hormone/growth factor/cytokine influences. In addition, a variety of functional studies demonstrate ECM-dependent cellular responses in vitro while genetic approaches clearly demonstrate critical requirements for genes encoding ECM components or modifiers of ECM, e.g., metalloproteases, in a wide variety of processes in vivo.

Immunohistochemical Study of Equine Endometrial Extracellular Matrix during the Oestrous Cycle

This study was designed to identify the distribution of type IV collagen, laminin, and fibronectin with an avidin-biotin method in sections of equine endometrial samples, fixed in Bouin's solution and embedded in paraffin wax. Thirty endometrial biopsies were collected at three different stages of the oestrous cycle. The basement membrane of luminal epithelium reacted positively with antibody against type IV collagen. Both type IV collagen and laminin were found in the basement membranes of endometrial glands, and fibronectin occurred diffusely in the interstitial tissue. Blood vessels expressed all of the extracellular matrix components studied. No differences in the distribution of extracellular matrix components were found at the different stages of sampling.

Distribution of fibronectin, laminin and collagen type IV in the materno-fetal boundary zone of the developing mouse placenta. Experimental study.

The objective of this study is to demonstrate the distribution of extracellular matrix components of fibronectin, laminin and collagen type IV in the materno-fetal boundary zone of the developing mouse placenta. Mice fetuses and placentas were removed serially every day until the 19th gestational day. Implantation sites were processed and stained by an immunohistochemical method by specific antiserums to fibronectin, laminin and collagen type IV. The distribution of the extracellular matrix components in cytotrophoblasts and giant cells of the developing mouse placenta were determined under light microscope. Fibronectin, laminin and collagen type IV immunostaining demonstrated a dynamic relationship changing day by day after the conception. At the 16th day cytotrophoblasts and giant cells were all positively stained by the extracellular matrix components. This study demonstrates that the regions of the developing mouse placenta produce specialized extracellular matrices which may contain different ratios of these polypeptides.

Fibronectin, laminin, and collagen IV as modulators of cell behavior during adrenal gland development in the human fetus.

The specific development of the human fetal adrenal gland requires cell proliferation, migration, apoptosis, and zone-specific steroidogenic activity. The present work was designed to determine the physiological significance of the previously identified spatial distribution of extracellular matrix components in the fetal gland. Primary cultures of human fetal adrenal cells grown on collagen IV, laminin, or fibronectin revealed that cell morphology was affected by environmental cues. Matrices also modulated the profile of steroid secretion by the fetal cells. Collagen IV favored cortisol secretion after ACTH or angiotensin II stimulation and increased dehydroepiandrosterone production when the AT(2) receptor of angiotensin II was specifically stimulated. These effects were correlated by changes in the mRNA levels of 3beta-hydroxysteroid dehydrogenase and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior. Collagen IV and laminin enhanced cell proliferation, and fibronectin increased cell death. This study is the first to demonstrate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.

Characterization and distribution of extracellular matrix components and receptors in GH 3B 6 prolactin cells

GH 3B 6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50–70 % of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH 3B 6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: α5β1 (FN receptor), α6β1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75 %) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH 3B 6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Immunohistochemical localization of collagen types I, III, and IV, laminin, fibronectin, and keratin in the endolymphatic sac.

We have employed immunohistochemistry to obtain baseline information on the molecular constituents of the extracellular matrix (ECM) of the endolymphatic duct (ED) and endolymphatic sac (ES) of the chinchilla. The results demonstrated that collagen types I and III were distributed in the subepithelial layer in the ED and ES, type IV collagen and laminin in the basement membranes, and fibronectin in the subepithelial layer and partly in the conglomerated cells in the ES. Collagen type III was diffusely distributed in the whole subepithelial layer of the ES, whereas collagen type I was concentrated densely in the deep layer of the interstitium, although gradually, the cuboidal epithelium in the ES was transformed into a flatter type in the ED. The epithelial cells of the ED and ES were clearly positive for keratin. This study deals, in particular, with the normal distribution of ECM components of the ED and ES of the chinchilla.

High-Resolution Ultrastructural Comparison of Renal Glomerular and Tubular Basement Membranes

Background/Aims: Glomerular basement membranes (GBM) and tubular basement membranes (TBM) consist of a fine meshwork composed mainly of type IV collagen. Each segment of tubules has specialized physiologic functions, and thus we investigated the ultrastructure of various basement membranes in rat kidneys. Methods: Since purifying basement membranes from different tubule segments is technically challenging, we employed tissue negative staining rather than conventional negative staining to compare the ultrastructures of proximal and distal TBM and GBM in normal rats. We also assessed the distribution of extracellular matrix components including type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes by immunohistochemistry. Results: TBM and GBM of normal rats showed a fine meshwork structure consisting of fibrils forming small round to oval pores. Short- and long-pore diameters in proximal tubules were 3.3 ± 0.5 and 3.9 ± 0.6 nm, respectively, and in distal tubules 3.5 ± 0.7 and 4.3 ± 0.8 nm, respectively. For GBM the respective diameters were 2.5 ± 0.5 and 3.0 ± 0.5 nm. Immunohistochemical analysis showed no significant difference in distribution of extracellular matrix components between proximal and distal TBM. However, immunofluorescence scores of α1 chain of type IV collagen, fibronectin, and laminin were higher in the TBM than in the GBM. On the other hand, heparan sulfate proteoglycan was higher in the GBM. Conclusion: Ultrastructural differences in renal basement membranes may be related to differences in physiologic function in each segment.