Function of latent TGFß binding protein 4 and fibulin 5 in elastogenesis and lung development.

Mice deficient in Latent TGFß Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFß2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFß1 or TGFß3 did not improve lung septation indicating that the TGFß isoform elevated in Ltbp4S(-/-) lungs is TGFß2. Expression of a form of Ltbp4 that could not bind latent TGFß did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFß complexes. Therefore, the change in TGFß-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFß2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.

Structural study of gubernaculum testis in fetuses with prune belly syndrome.

PURPOSE: We compared and contrasted the structure of the gubernaculum testis in fetuses with prune belly syndrome and normal controls. MATERIALS AND METHODS: We studied a total of 6 gubernacula from 3 male fetuses with prune belly syndrome and a total of 14 from 7 male fetuses without an anomaly. Gubernacular specimens were cut into 5 µm sections and stained with Masson trichrome to quantify connective tissue and smooth muscle cells, with Weigert stain to observe elastic fibers and with picrosirius red with polarization to observe collagen. Immunohistochemical analysis was done with tubulin to observe the nerves. Images were captured with a BX51 microscope and DP70 camera (Olympus®). Stereological analysis was done with Image-Pro and ImageJ (MediaCybernetics®) using a grid to determine volumetric density. Means were statistically compared with the Mann-Whitney test. All tests were 2-sided with p <0.05 considered statistically significant. RESULTS: Prune belly syndrome fetuses were at 17 to 31 weeks of gestation and control fetuses were at 12 to 35 weeks of gestation. Quantitative analysis showed no difference in the volumetric density of smooth muscle cells in prune belly syndrome vs control gubernacula (mean 15.70% vs 19%, p = 0.2321). Collagen fiber analysis revealed a predominance of green areas in prune belly syndrome gubernacula, suggesting collagen type III, and a predominance of red areas in control gubernacula, suggesting collagen type I. Elastic fibers were significantly smaller in prune belly syndrome gubernacula than in control gubernacula (mean 14.06% vs 24.6%, p = 0.0190). Quantitative analysis demonstrated no difference in the volumetric density of nerves in prune belly syndrome or control gubernacula (mean 5.200% vs 3.158%, p = 0.2302). CONCLUSIONS: The gubernaculum in fetuses with prune belly syndrome had altered concentrations of collagen and elastic fibers. These structural alterations could be one of the factors involved in cryptorchidism in prune belly syndrome.

Study of the ureter structure in anencephalic fetuses.

PURPOSE: The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. MATERIALS AND METHODS: We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). RESULTS: The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. CONCLUSIONS: Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate that anencephalic fetal ureters tend to have significant structural alterations, probably due to cerebral lesions with consequent brain control damage of ureter nerves.

Identification of a Primary Stroma and Novel Endothelial Cell Projections in the Developing Human Cornea.

Purpose: To investigate the initial events in the development of the human cornea, focusing on cell migration, and extracellular matrix synthesis and organization. To determine whether elastic fibers are present in the extracellular matrix during early human corneal development. Methods: Human corneas were collected from week 7 to week 17 of development. An elastic fiber-enhancing stain, tannic acid-uranyl acetate, was applied to all tissue. Three-dimensional serial block-face scanning electron microscopy combined with conventional transmission electron microscopy was used to analyze the corneal stroma. Results: An acellular collagenous primary stroma with an orthogonal arrangement of fibrils was identified in the central cornea from week 7 of corneal development. At week 7.5, mesenchymal cells migrated toward the central cornea and associated with the acellular collagenous matrix. Novel cell extensions from the endothelium were identified. Elastic fibers were found concentrated in the posterior peripheral corneal stroma from week 12 of corneal development. Conclusions: This study provides novel evidence of an acellular primary stroma in the early development of the embryonic human cornea. Cell extensions exist as part of a communication system and are hypothesized to assist in the migration of the mesenchymal cells and the development of the mature cornea. Elastic fibers identified in early corneal development may play an important role in establishing corneal shape.

Absence of TGFbeta signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression, impaired elastogenesis, and aneurysm.

To address the requirement for TGFbeta signaling in the formation and maintenance of the vascular matrix, we employed lineage-specific mutation of the type II TGFbeta receptor gene (Tgfbr2) in vascular smooth muscle precursors in mice. In both neural crest- and mesoderm-derived smooth muscle, absence of TGFbeta receptor function resulted in a poorly organized vascular elastic matrix in late-stage embryos which was prone to dilation and aneurysm. This defect represents a failure to initiate formation of the elastic matrix, rather than a failure to maintain a preexisting matrix. In mutant tissue, lysyl oxidase expression was substantially reduced, which may contribute to the observed pathology.

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study.

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

The connective tissue of the adductor canal--a morphological study in fetal and adult specimens.

The adductor canal is a conical or pyramid-shaped pathway that contains the femoral vessels, saphenous nerve and a varying amount of fibrous tissue. It is involved in adductor canal syndrome, a claudication syndrome involving young individuals. Our objective was to study modifications induced by aging on the connective tissue and to correlate them to the proposed pathophysiological mechanism. The bilateral adductor canals and femoral vessels of four adult and five fetal specimens were removed en bloc and analyzed. Sections 12 microm thick were obtained and the connective tissue studied with Sirius Red, Verhoeff, Weigert and Azo stains. Scanning electron microscopy (SEM) photomicrographs of the surfaces of each adductor canal were also analyzed. Findings were homogeneous inside each group. The connective tissue of the canal was continuous with the outer layer of the vessels in both groups. The pattern of concentric, thick collagen type I bundles in fetal specimens was replaced by a diffuse network of compact collagen bundles with several transversal fibers and an impressive content of collagen III fibers. Elastic fibers in adults were not concentrated in the thick bundles but dispersed in line with the transversal fiber system. A dynamic compression mechanism with or without an evident constricting fibrous band has been proposed previously for adductor canal syndrome, possibly involving the connective tissue inside the canal. The vessels may not slide freely during movement. These age-related modifications in normal individuals may represent necessary conditions for this syndrome to develop.

Lorenzo Gotte (1926-1991): a pioneer of elastin.

Lorenzo Gotte (1926-1991) was an outstanding histologist at the School of Medicine of Padua. This year marks the 25th anniversary of his passing away - commemorated during the recent congress of the Italian Society for the Connective Tissue (SISC), held in Padua (September 30 - October 1, 2016). This brief note recalls this outstanding figure: indeed, forthose who knew him, Lorenzo Gotte was an exceptional scientist and at the same time, an unparalleled teacher - and, for many, a great friend. It is still difficult to separate these aspects of his personality, so intertwined in his life: studying elastin and elastic tissue was a passion central to Gotte's life.

The tissue distribution of murine Abcc6 (Mrp6) during embryogenesis indicates that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.

Mutations in the gene coding for the ABC transporter, ABCC6, in humans cause Pseudoxanthoma elasticum, which is characterized by the deposition of aberrant elastic fibers. To investigate whether the presence of ABCC6 in tissues synthesizing elastin is required for elastin deposition and elastic fiber assembly, we have compared the steady-state levels and tissue distribution of Abcc6 and tropoelastin mRNAs during mouse embryogenesis. Whereas tropoelastin mRNA levels rose during embryogenesis and were the highest in neonatal mice, Abcc6 mRNA levels remained constantly low throughout embryogenesis. In some tissues, both Abcc6 and tropoelastin mRNA were detected. However, Abcc6 mRNA and protein were not detected in neonatal aorta and arteries, which produce large amounts of elastin indicating that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.

Elastic fibers in fetal dermis.

Dermal elastic tissue in fetal skin was evaluated by light microscopy using three different staining techniques. Postmortem skin was obtained from the abdominal wall of 45 fetuses ranging in age from 8 to 42 weeks. Elastic fibers were first detected in skin from fetuses of 22 weeks and, with increasing gestational age, appeared to increase in quantity and complexity. Skin from fetuses older than 32 weeks had a well-developed network of elastic fibers throughout the dermis. There was no diminution of dermal elastic tissue during the latter part of the sixth lunar month as had been reported in a previous study.

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues.

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.

Expression of collagens and decorin during aortic arch artery development: implications for matrix pattern formation.

The elastic matrix of the large arteries shows a high level of spatial order. However, the mechanisms by which such order is established and maintained are largely unknown. The embryonic development of the avian heart and great vessels provides an appropriate model to investigate these mechanisms. In control embryos, an elastic matrix with a high level of spatial order develops in the nascent great vessels. But after the normal vascular smooth muscle (VSM) progenitor cells in the great vessels are experimentally replaced by other VSM progenitor cells, the elastic extracellular matrix is congenitally disordered. The present study used this model to test the hypothesis that the proteoglycan decorin was involved in the establishment and maintenance of the normal three-dimensional spatial order of the vascular elastic matrix. The temporospatial expression of decorin was analysed during development of normal vessels and in experimental vessels with surrogate VSM. The results showed the following: (1) the expression of decorin was related in time and space to the establishment of large helical collagen type III fibers that are characteristic of the normal elastic extracellular matrix; (2) in the experimental extracellular matrix there were few helical fibers of collagen type III, but those that were present remained positive for decorin; and (3) in both control and experimental vessels, decorin associated with neither fibers of collagen type I nor fibers of collagen type III in any conformation other than the large helical fibers. These data indicate a previously unrecognized relationship between decorin and the spatial order of the physiologically significant helical fibers of collagen type III.

An electron microscopic study on the type I pneumocyte in the cat: pre-natal morphogenesis.

This investigation describes the pre-natal morphogenesis of the type I pneumocyte subsequent to its differentiation from pulmonary epithelium. Cells lining subpleural alveolar septa were photographed from serial sections with the electron microscope, and a three-dimensional representation of each cell was obtained by transferring the contours of the cell membranes from montages to transparent plastic sheets which were then spaced to scale and stacked. The results of this study indicate that: The nascent blood-air barrier of a 50-day reconstructed cell was twice as thick as the average definitive barrier; definitive barrier thickness was observed in some areas in a 63-day reconstructed cell; the amorphous component of elastic tissue which appears peripherally in septal connective tissue during pre-natal morphogenesis may be directly juxtaposed to the basal lamina of the alveolar epithelium; the orientation of the cell junction between a pneumocyte and its neighboring cells, as observed in sections of alveolar septa, changes as the contour of the pneumocyte changes from simple abutment to overlapping patterns.

The development of fibrocartilage in the elastic tendon of the chicken wing.

The elastic tendon of the chicken wing has five morphologically distinct regions. One of these regions is a distally located fibrocartilage from which fibrous connections extend to the capsule of the distal radius. In adult birds, this region shows the characteristics of a tendon-compressed fibrocartilage, with an accumulation of proteoglycans between thick collagen bundles arranged in a basket-weave formation. Here we study the development of this fibrocartilage in order to of compare it with other tendon fibrocartilages and try to identify the factors involved in fibrocartilage differentiation. This fibrocartilage initially developed by cell enlargement and accumulation of vimentin, with simultaneous deposition of proteoglycans in the extracellular matrix and an increase in the amount and thickness of collagen bundles. Elastic fibers were minor components associated with the collagen bundles. Cells could be classified into two main types. One was typically fibrocartilaginous and the other was fibroblast-like, the latter occurring in close association with the collagen bundles. These results establish the steps in the development of the elastic tendon fibrocartilage and provide a basis for future studies.

Increase of segments of elastic-type blood vessel walls in fetal placental stem villi during pre-eclampsia at term.

In recent studies we described the presence of elastic-type blood vessels within trunci and rami chorii of human placental stem villi. For systemic and pulmonary hypertension it is known that elastic fibres are enhanced in arteries. The aim of our study was, therefore, to examine whether pre-eclampsia may lead to an increase of elastic tissue fibres in blood vessel walls of placental stem villi and whether there are differences in the thickness of blood vessel walls within these villi when compared to normotensive pregnant women. Twenty-six women with uncomplicated pregnancies and 25 patients with pre-eclampsia were investigated. Unfixed cryostat serial sections were processed for conventional orcein staining and for the demonstration of alpha-actin-immunoreactivity. The intensity of orcein staining of stem villus blood vessel walls was evaluated by a semiquantitative score method. Significant higher intensities of orcein staining (P<0.00001) were calculated for blood vessel walls of placentae with pre-eclampsia. The amount of thick stem villus vessels (>41 microm) increased during pre-eclampsia from 39 gestational weeks onwards. Our study demonstrates that segments of thick blood vessel walls and elastic-type vessel walls are increased in placental stem villi of patients with pre-eclampsia. This reaction may protect the fetal placental vessels and avert an increase of the fetal hypertension.

Modifications of erectile tissue components in the penis during the fetal period.

BACKGROUND: The penile erectile tissue has a complex microscopic anatomy with important functions in the mechanism of penile erection. The knowledge of such structures is necessary for understanding the normal physiology of the adult penis. Therefore, it is important to know the changes of these penile structures during fetal development. This study aims to analyze the development of the main components of the erectile tissue, such as collagen, smooth muscle fibers and elastic system fibers, in human fetuses. METHODOLOGY/PRINCIPAL FINDINGS: We studied the penises of 56 human fetuses aged 13 to 36 weeks post-conception (WPC). We used histochemical and immunohistochemical staining, as well as morphometric techniques to analyze the collagen, smooth muscle fibers and elastic system fibers in the corpus cavernosum and in the corpus spongiosum. These elements were identified and quantified as percentage by using the Image J software (NIH, Bethesda, USA). From 13 to 36 WPC, in the corpus cavernosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 19.88% to 36.60%, from 4.39% to 29.76% and from 1.91% to 8.92%, respectively. In the corpus spongiosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 34.65% to 45.89%, from 0.60% to 11.90% and from 3.22% to 11.93%, respectively. CONCLUSIONS: We found strong correlation between the elements analyzed with fetal age, both in corpus cavernosum and corpus spongiosum. The growth rate of these elements was more intense during the second trimester (13 to 24 WPC) of gestation, both in corpus cavernosum and in corpus spongiosum. There is greater proportional amount of collagen in the corpus spongiosum than in corpus cavernosum during all fetal period. In the corpus spongiosum, there is about four times more collagen than smooth muscle fibers and elastic system fibers, during all fetal period studied.

Elastogenic protein expression of a highly elastic murine spinal ligament: the ligamentum flavum.

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues.

Development of collagen fibres and lysyl oxidase expression in the presumptive dermis of chick limb bud.

Lysyl oxidase (LOX) plays a critical role in the formation of cross-linkages in extracellular matrix molecules. Thus, it is essential for the biogenesis and homeostasis of the connective tissue matrix. During development, collagen fibres and elastic system fibres emerge and accumulate in a temporospatial manner in the presumptive dermis of chicks. In this study, we investigated LOX mRNA expression by laser capture microdissection and RT-qPCR and LOX protein localization by immunohistochemistry. The picrosirius polarization method was used to investigate a relation between collagen accumulation and LOX expression. PCR analysis showed that the expression of LOX mRNA in the presumptive dermis became apparent at embryonic day 13 and increased considerably by ED17. Immunohistochemical staining for LOX in the dermis was very low at all stages of development. Accumulation of collagen fibres was seen in the dermis on ED10, and higher wavelengths of birefringence became evident by ED13. Our findings suggest that the temporal pattern of LOX mRNA expression correlates with collagen fibre accumulation in the dermis of the developing chick limb bud, whereas LOX expression was relatively constant at the protein level.