Expression of type VIII collagen during morphogenesis of the chicken and mouse heart.

The expression of type VIII collagen is restricted, in adult mammals, to specialized extracellular matrices and to a select subset of blood vessels. We have examined the distribution of type VIII collagen in sequential stages of mouse and chicken embryos and found a temporal and spatially restricted pattern of expression during cardiogenesis. Type VIII collagen was first detected by immunocytochemistry on Day 11 in the developing mouse embryo and at stage 19 in the chicken embryo. The distribution of this protein was rapidly modulated during cardiac morphogenesis. Initially (Day 11 in the mouse embryo), type VIII collagen was associated with cardiac myoblasts. From Days 15 to 18, the immunoreactive component was progressively diminished in the myocardium; however, this collagen was observed in the subendocardial layer of the atrioventricular canal and later in the cardiac jelly (or the myocardial basement membrane, an area associated with the formation of cardiac valves). On Day 17, type VIII collagen was also detected in the subendothelium (intima) and tunica media of large vessels. Neonatal and adult hearts contained low to undetectable levels of type VIII collagen. The presence of type VIII collagen was confirmed by immunoblot analysis of heart extracts at different stages of development. A major 185-kDa component, as well as polypeptides of 68 and 15 kDa, reacted with anti-type VIII collagen IgG. Exposure of heart extracts to hyaluronidase or reducing agent eliminated immunoreactivity of the 185-kDa component but not that of the 68- and 15-kDa polypeptides. Type VIII collagen therefore appears to be associated with a hyaluronidase-sensitive component of the extracellular matrix during a temporally restricted stage of embryonic cardiogenesis. The contribution of this collagen to cardiac morphogenesis might reside, in part, in its ability to influence the differentiation of the myocardium and formation of the cardiac valves.     

Expression and localization of laminin 5, laminin 10, type IV collagen,and amelotin in adult murine gingiva

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules—laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)—and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.     

Cardiac jelly arrangement during the formation of the tubular heart of the chick embryo.

The hearts of chick embryos from stages 9 to 11 according to Hamburger and Hamilton are studied by means of the electron microscopy, and the arrangement of the cardiac jelly (CJ) during the different steps of the fusion of the paired heart anlage is described. It is shown that two different areas can be distinguished in CJ: the CJ located between the endocardium and the myocardium (MECJ) and the CJ located between the endocardium and the ventral foregut endoderm (EECJ). MECJ is a zone poor on ultrastructural components most of which are unbanded filamentous material and low-density amorphous material, and its arrangement remains constant during the whole fusion process; EECJ, on the contrary, is very rich in ultrastructural components containing greater amounts of high-density amorphous material, collagen fibrils and cellular, detritus, and the arragement of this area of the CJ undergoes changes during the different fusion steps. Alcian blue staining does not show difference between both areas of CJ. It is suggested that the CJ can play a role during the fusion of the heart. In addition, some observations are reported which suggest that all the epithelial tissues surrounding the CJ can take part in the elaboration of that extracellular material.     

Ultrastructural study of long spacing collagen fibres and basal lamina in malignant schwannoma

It was found by electron microscopy that extracellular darkly stained materials (DSM) observed abundantly in a case of malignant schwannoma were closely related to both basal lamina and fibrous long spacing collagen (FLS). The FLS were characterized by the cross bands with a 95 nm periodicity, and longitudinally aligned filaments, 9 nm in diameter, while DSM consisted of amorphous material, and 9 nm filaments. The filaments in DSM and FLS were similar in diameter and morphology to reticular fibres in basal laminae. The DSM were continuous with both dark bands of FLS and basal laminae. These results indicate that basal laminae may be the common origin of DSM and FLS. Ultrastructural features of longitudinal, transverse and oblique sections were described.     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles 相似文献   

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50–800 μg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H₂O₂, 50–200 μM), or tumor necrosis factor α (TNFα, 40 ng/ml) for 24–48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [³H]-thymidine or [³H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 μg/ml) and double therapeutic (100 μg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200–800 μg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 μg/ml) significantly inhibited TNFα-induced apoptosis in myocytes, with 12.3±1.46% cells being apoptosis in CP treatment group and 37.0±7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by ～50% and ～90% reduction of AII-induced collagen synthesis at 50 and 100 μg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.     

Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Exogenous hydrogen sulfide attenuates diabetic myocardial injury through cardiac mitochondrial protection

In the study, we investigated how exogenous H₂S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50?mg/kg) and the daily administration of 100?μM NaHS (sodium hydrosulfide, an H₂S donor) in the diabetes?+?NaHS treatment group. At the end of 4, 8, and 12?weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H₂S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.     

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

Accumulation of components of basal laminae: association with the failure of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice

Aganglionosis occurs in the terminal colon of the ls/ls mouse because an intrinsic defect of the presumptive aganglionic tissue prevents the entry and colonization of this portion of the bowel by migrating neural crest cells. The current study was undertaken to determine if abnormalities of the extracellular matrix could be identified in this segment that might account for migratory failure. Since basal laminae of the muscularis mucosa are overproduced in the aganglionic segment of adult ls/ls mice, we examined components of basal laminae in fetal gut from Day E 11 to Day E 16 of gestation. This period spans the time of enteric ganglion formation. Laminin and collagen type IV were studied by immunocytochemistry and proteoglycans by staining glycosaminoglycans with Alcian blue. Abnormalities of each of these components occur during development of the presumptive aganglionic bowel in the ls/ls mouse and could be detected as early as Day E 11. These defects consist mainly of an overabundance of these materials, both in defined basal laminae and throughout the extracellular space of the mesenchyme. Electron microscopic observations in the presumptive aganglionic ls/ls colon revealed a thickening of basal laminae and exceptionally wide intercellular spaces between smooth muscle myoblasts that contained an irregular fibrillar material, consisting of 4.5- to 6.0-nm filaments associated with 14- to 20-nm granules. Fibrillar and flocculant material was continuous with formed basal laminae, and was concentrated in the same areas found to have an overabundance of laminin immunoreactivity. These observations indicate that there is an accumulation of extracellular matrix material, including components of basal laminae, that (i) precedes the formation of enteric ganglia, (ii) is in the path through which enteric neural precursors from the crest would have to migrate, and (iii) is limited to the aganglionic and hypoganglionic ls/ls bowel. These data are consistent with the hypothesis that components of basal laminae contribute to the inability of crest cells to colonize the terminal bowel of ls/ls mice.     

Cardiac myofibrillogenesis inside intact embryonic hearts

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 μm filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.     

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.     

X-ray diffraction analysis of the 3D organization of collagen fibrils in the wall of the dogfish egg case

The wall of the egg case of the dogfish,Scyliorhinus canicula, contains a network-forming collagen assembled into a regular three-dimensional (3D) structure. It accomplishes supportive, protective and filtering functions for the embryo contained within it. The collagen molecules in the egg case are organized into a body-centred unit cell of dimensions (mean ± s.d.) (11.6 ± 1.0) nm X (11.6 ± 1.0) nm X (81.6 ± 3.2) nm, which belongs to the I422 space group. At a higher hierarchical level, the collagen molecules assemble into parallel arrays of fibrils, ca. 100 nm in diameter, which aggregate to form laminae ca. 0.5 μm thick. These laminae are organized into a plywood-like structure and account for 98% of the thickness of the wall of the egg case. X-ray diffraction patterns of the wall of the egg case were taken along mutually perpendicular directions, one being perpendicular to the surface of the egg case. Three different kinds of diffraction pattern were observed. One of the patterns was characteristic of an X-ray direction perpendicular to the laminae in the egg case (along the x-direction). The two other patterns were obtained with the X-rays directed parallel to the plane of the laminae, either along the capsule long axis (z) or perpendicular to this (y). These two patterns were observed interchangeably in either of the x- or y-directions depending on the specimen. The diffraction patterns were analysed and interpreted taking into consideration the 3D electron microscope data of the egg case. The results confirm and extend previous findings from transmission electron microscopy and low-angle X-ray diffraction and they suggest that there is only one major type of ordered collagen arrangement in the wall of the egg case.