Expression of basement membrane type IV collagen chains during postnatal development in the murine cochlea

An immunofluorescence study was performed to examine the temporal and spatial patterns of expression for the different type IV collagen chains during postnatal cochlear development. At birth, the classical chains (4A1 and 4A2) were widely expressed, while the novel chains (4A3, 4A4, and 4A5) were completely absent. Activation of the novel chains was observed at 4 days of age, with intense, widely distributed immunostaining suggesting that most of the cells in the cochlea express the novel chains at this developmental stage. From day 8 through day 14, developmental inactivation of the novel chains results in a reduction of generalized immunoreactivity with a concomitant elevation of specific staining in the membranous structures bounding the interdental cells of the spiral limbus, the inner sulcus, the basilar membrane, and in a fibrous bed of staining radiating from the spiral prominence into the region of the spiral ligament which corresponds to the location of the root cell processes. This pattern of intense immunostaining for the novel chains persists through adulthood. The classical chains are expressed in these same anatomical regions only transiently (from day 6 to day 10), after which a gradual developmental inactivation leads to the adult expression pattern where classical collagen chains are found primarily in the perineurium, in the membranes surrounding the spiral ganglion cell bodies, and in the vascular basement membranes of the spiral ligament and the stria vascularis. The complex developmental pattern of expression for the type IV collagen chains in the murine cochlea is similar to that observed in the murine kidney, which is the other major site for basement membrane pathology in Alport syndrome.     

Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family

Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (V. vulnificus protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.     

Changes in cochlear function after double-membrane rupture in the guinea pig

We measured the transiently evoked otoacoustic emissions (TEOAEs), compound action potentials (CAPs) and cochlear microphonics (CMs) in guinea pigs after rupture of the round window membrane alone (n = 5) or of the round window membrane with localized cochlear damage (n = 10). The localized cochlear damage entailed rupture of Reissner's membrane with damage to the stria vascularis. We determined the time course of changes in the total echo power (TEP) in TEOAEs and the minimal detectable levels of CAPs and CMs. The endocochlear potential (EP) was measured in the cochlea with localized damage. There were no changes in TEOAEs, CAPs or CMs in the guinea pigs subjected to round window membrane rupture alone, but the minimal detectable levels of CAPs and CMs were increased in all the guinea pigs in which TEOAEs were absent after rupture of the round window membrane with localized cochlear damage. Our results suggest that double-membrane rupture (rupture of the round window membrane with localized cochlear damage) produces acute sensorineural hearing loss. The hearing loss appeared to be related to damage to the cochlea, which may be induced by influx of potassium-rich endolymph into the perilymph, and by morphological damage to the scala media.     

Quantitative analysis of Na+ and Ca2+ current contributions on spike initiation in the newt olfactory receptor cell

The developmental localization patterns of collagen type IV alpha1-5 chains, laminin-1, laminin-5, and laminin alpha2 chain were analyzed in the embryonic mouse eye using isoform specific antibodies and immunofluorescence microscopy. Laminin-1 isoform and alpha1-2(IV) were ubiquitously expressed along the ocular surface basement membranes at a very early stage of eye development. Alpha3-5(IV) were first detected at later stages of development, and exhibited a variable distribution pattern along the ocular surface basement membrane. In contrast, expression of the laminin alpha2 chain was restricted to the conjunctival basement membrane, and was first detected during the same developmental period in which keratin K4-positive, differentiated conjunctival epithelial cells were observed. Although laminin-5 was uniformly expressed along the adult ocular surface basement membrane, during embryogenesis it was first incorporated into the conjunctival basement membrane structure. These data suggest that some of the laminin isoforms, including laminin alpha2 and laminin-5, may play a role in the formation of a conjunctival-type basement membrane. The temporal relationship between the localization of these molecules to the conjunctival basement membrane and the appearance of differentiated conjunctival epithelial cells suggests a role for external influence on the differentiation pathways of ocular surface epithelium.     

Platelet-derived growth factor-stimulated secretion of basement membrane proteins by skeletal muscle occurs by tyrosine kinase-dependent and -independent pathways

The basement membrane of skeletal muscle is produced by the muscle cells it ensheathes and by nonmuscle cells located in the surrounding extracellular matrix. In this study, we have shown that platelet-derived growth factor (PDGF) stimulates secretion of three basement membrane components of skeletal muscle: laminin (70% increase), fibronectin (30%), and type IV collagen (70%). Furthermore, we have found using the signal transduction inhibitors, genistein (tyrosine kinase inhibitor), phorbol 12-myristate 13-acetate (protein kinase C (PKC) inhibitor), thapsigargin (depletes intracellular Ca2+ stores), and H89 (protein kinase A inhibitor), that PDGF-stimulated secretion of these proteins occurs through distinct signaling pathways. Densitometry of Western blots of L6 myoblast supernatant indicates that the PDGF-induced increase in secretion of laminin and type IV collagen is tyrosine kinase-dependent. The increase in type IV collagen secretion also shows dependence on PKC, as well as the release of intracellular Ca2+. Inhibition of either of these pathways reduces the increase in type IV collagen secretion to 20%. In contrast, the PDGF-induced increase in laminin secretion is unaffected by inhibition of either PKC or intracellular Ca2+ release. The increase in fibronectin secretion by PDGF uses yet a third set of signals. PDGF-induced fibronectin secretion is not dependent on tyrosine kinase activity but is dependent on protein kinase A as well as the release of intracellular Ca2+. These divergent signaling pathways provide for independent regulation of basement membrane protein secretion, allowing a muscle cell to modify both the quantity and composition of its basement membrane in response to its environment.     

Corneal epithelial basement membrane after experimental anterior stromal puncture in guinea pigs: immunohistochemical study

We investigated alterations in the immunolocalization of the components of epithelial basement membrane (BM), type IV collagen, and laminin in guinea pigs subjected to anterior stromal puncture (ASP) of the cornea performed with a standardized needle. Localization of BM components beneath the corneal epithelium was indicated by laminin immunoreactivity. The BM was interrupted by needle punctures immediately after ASP. During healing, type IV collagen immunoreactivity was detected transiently in the BM of some of the ASP-treated corneas, but no reactivity was observed in normal epithelial BM. Development of type IV collagen immunoreactivity was probably caused by an alteration of the alpha-chains or by an unmasking of the antigenicity of this collagen type, which may be related to an increase in the adhesiveness of the epithelium following ASP.     

Use of extracorporeal life support for adult patients with respiratory failure and sepsis

Na+,K+-ATPase activity is abundant on the basolateral infoldings of the strial marginal cells and contributes to the maintenance of the characteristic electrolyte composition of the endolymph. However, the stria vascularis of the cochlea is known not to be innervated. In order to clarify its humoral regulation by serotonin, the K+-p-nitrophenylphosphatase activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and in guinea pigs treated with reserpine, 5-hydroxytryptamine or reserpine plus 5-hydroxytryptamine. K+-p-nitrophenylphosphatase activity was almost completely depressed 3-20 days after reserpine administration. Ten days after reserpinization, followed by repeated 5-hydroxytryptamine treatment, the enzyme activity was detectable. These results suggest that 5-hydroxytryptamine increases the phosphatase activity. Thus, the function of the stria vascularis in producing cochlear endolymph may be regulated by 5-hydroxytryptamine.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 microg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 microm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 microg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along the dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Alterations in the distributions of the basement membrane components after secondary inner ear immune reaction in guinea pigs

Alterations in the distributions of type IV collagen (C-IV), laminin (La), and fibronectin (Fib), which are important components of the basement membrane, in the inner ear following secondary endolymphatic sac immune response were studied immunohistologically using control animals for comparison. Endolymphatic hydrops developed following direct secondary keyhole limpet hemocyanin (KLH) challenge to the endolymphatic sac in systematically pre-sensitized animals. In the endolymphatic sacs of control animals, C-IV and La were localized continuously just under epithelial cells whereas Fib was present intermittently in subepithelial connective tissue. In the endolymphatic sac, following secondary KLH challenge, linear subepithelial localizations of C-IV and La were interrupted, thinner and more loosely aggregated with numerous inflammatory cellular infiltrates on days 2-4. Following these changes, endolymphatic hydrops in the cochlea developed and peaked on days 4-7. On days 1-7, Fib was strongly but sporadically localized in subepithelial cells. These results suggest that C-IV and La may play important roles in the regulation of endolymph whereas Fib may be related to the restoration of injured endolymphatic sac tissue in animals exposed to a secondary challenge.     

Profile of the Institute for Rehabilitation and Research

Indirect immunofluorescence was used for the localization of the primary collagens, fibronectin and laminin. Specimens were extracted from untreated teeth with periapical lesions from patients 20 to 30 years of age. An histological examination enabled the differentiation of granulomas and cysts, and 5 microns sections were used for the indirect immunofluorescence procedure. Antibodies against Type I, Type III, and Type V collagen and for fibronectin and laminin were obtained from glycoproteins of human cells. The antibody against Type IV collagen was prepared from Type IV collagen of beef retina. All the glycoproteins investigated were expressed in apical lesions. The intensity of the immunostaining appeared more positive at the external area compared with the center of the lesion. The type IV collagen was specific for the basement membrane of cysts. The immunofluorescence reactions of fibronectin and of laminin were similar in intensity in both granulomas and cysts.     

Rapid expression and specific localization of tenascin in gastric ulcer healing in rats

This study was done to investigate the gene expression and localization of tenascin in ulcerated gastric tissues during the healing process with Northern blot analysis and immunohistochemical technique. Gastric ulcers in rats were produced by acetic acid. Tenascin mRNA levels in the ulcerated tissue were significantly increased in a biphasic manner (12 h and day 5), preceding the increase in collagen type IV and laminin mRNA levels, and returned to control levels on day 11. In intact tissues, tenascin was mainly localized in the basement membrane above the proliferative zone, in contrast to the predominant localization of collagen type IV and laminin below the proliferative zone. On the ulcer margin from 12 h to day 5, tenascin was abundantly observed in the lamina propria around nonproliferating new epithelial cells, but collagen type IV and laminin were not seen in this lamina propria. On day 7, tenascin, expressed in the lamina propria, was replaced by collagen type IV and laminin. Thus, the rapid expression and unique localization of tenascin suggest the important role of tenascin in gastric ulcer healing.     

Longitudinal endolymph movements induced by perilymphatic injections

Endolymph movements and endocochlear potential (EP) changes were measured during disturbances of perilymphatic pressure. induced by injecting artificial perilymph into scala tympani (ST) or scala vestibuli (SV) of the guinea pig cochlea. Injections were performed either with or without an outlet made in the opposite perilymphatic scala. Injections into ST without an outlet induced large pressure changes but virtually no endolymph movement or EP change. Injection at the same rate into ST with an outlet in SV produced smaller pressure changes which were accompanied by a basally-directed displacement of endolymph and significant EP changes. The magnitude of endolymph displacements and EP changes varied as a function of injection rate. Injections into SV, either with or without an outlet in ST, produced apically-directed endolymph displacement and EP changes. For the SV injections without an outlet, the cochlear aqueduct and round window are likely to provide an outlet and compliance, permitting flow along the perilymphatic scalae to occur even when no ST outlet was provided. We conclude that endolymph movements are not dependent on the absolute pressure of the perilymph, but instead occur when small, sustained pressure gradients are present across the cochlear partition, corresponding to times when perilymph flow is induced. This study demonstrates that in the normal. sealed cochlea, endolymph and EP are insensitive to fluid injections into ST, but are sensitive to fluid injections into SV. Endolymph movements are therefore unlikely to be generated by cerebrospinal fluid pressure fluctuations (such as those produced by respiration, posture changes, coughing, sneezing, etc) which are transmitted to ST by the cochlear aqueduct.     

Inositol 1,4,5-trisphosphate receptor expression in odontoblast cells

Although the exact pathogenesis of mustard gas-induced dermal toxicity remains elusive, morphopathological data gathered in controlled animal and in vitro investigations is providing important clues as to approximate mechanisms. Our laboratory has been studying dermal effects of the chemical warfare agent, sulfur mustard, in a variety of animal models, cultured isolated human cells, and in vitro organotypic skin models. Published anatomical, pathological, and ultrastructural results of these studies have documented consistent cellular and basement membrane zone effects irrespective of the model. Cellular effects include the early targeting of basal cells of the stratum basale to the exclusion of other epidermal cells, with nuclear and cytoplasmic indications of cell injury and cell death. Effects on the basement membrane zone include the formation of characteristic microvesicles in the lamina lucida of those models which possessed structural components of a true basement membrane. We are now investigating effects on proteins of the basement membrane microenvironment and correlate in the present paper the morphopathology of sulfur mustard dermal lesions with immunohistochemical study of bullous pemphigoid antigen, laminin, type IV collagen, and type VII collagen.     

Studies with recombinant proteins of Ehrlichia risticii: identification of strain-specific antigen as a protective antigen

Reconstitution of basement membrane structures after "sandwich-technique" grafting of severe deep burns is demonstrated with use of immunohistochemical techniques. Cryosections of human skin after epifascial burn wound excision and sandwich grafting were stained with monoclonal antibodies against type IV and VII collagen, polyvalent antiserum against type VI collagen, and polyvalent antibody against laminin. Standard hematoxylin and eosin histologies were performed for morphologic correlation. Reorganization of the mesenchymal border zone (basement membrane), after transplantation of extremely expanded split-thickness skin autografts overlaid with glycerolized split-thickness skin allografts onto debrided human full-thickness wounds, occurred from day 5 to day 35. The autografts reepithelize the spaces between the mesh structure, which has been covered primarily exclusively with allogenic skin, and form a layered squamous epithelium, with an underlying three-dimensional basket-weave array of collagen in the remodeled neodermis after epifascial excision. Immunochemical techniques detect the reconstitution of a basement membrane zone with a typical architecture and distribution of laminin, type IV, and type VII collagen being built up 1 week to 5 weeks after sandwich grafting. These structures can be seen in the autografts during the first 2 weeks and are consistent in the whole reconstituted skin after day 35. To our knowledge this is the first report of the expression of type VI collagen in these types of wounds. The findings are compared with the expression of type VI collagen in healthy skin. The results indicate that the modified sandwich-grafting technique is an adequate means for early burn wound closure and resurfacing of third-degree burn wounds and leads to the reconstitution of dermal qualities.     

Infections of the hand

The key event associated with the initiation of angiogenesis is the localized degradation of the vascular basement membrane. Because of its complex structure, any remodelling and/or modification of the basement membrane must involve the co-ordinated function of a number of different enzyme systems. Type IV collagen is a major protein component (60-90%) of the basement membrane and its degradation is crucial to the initiation of angiogenesis. This study has focused on the mechanisms by which C6 astrocytoma cells degrade human type IV collagen. C6 astrocytoma cells use components of two major degradative pathways to degrade collagen type IV. The major matrix metalloproteinase identified is the activated form (68-KDa) of gelatinase A (72-KDa matrix metalloproteinase) and a serine sensitive 1000-KDa collagenase type IV degrading activity which appears to have the characteristics of a novel extracellular proteasome.     

Immunohistochemical localization of extracellular matrix proteins in luteal phase endometrium of fertile and infertile patients

The lack of expression of certain components involved in cell adhesion and migration is believed to contribute to endometrial dysfunction and implantation failure. The purpose of this study was to investigate whether luteal phase endometrium in women with unexplained infertility differs, with respect to specific extracellular matrix (ECM) proteins, from endometrium of normal fertile women. A panel of monoclonal antibodies to collagen type IV, fibronectin and laminin was used to characterize the localization of ECM components in the different endometrial compartments. Precisely timed endometrial biopsies obtained at 4, 7, 10 and 13 days following the luteinizing hormone surge were obtained from 22 normal fertile women (group 1) and 24 women suffering from unexplained infertility (group 2). Paraffin-embedded sections were labelled using the streptavidin-biotin alkaline phosphatase technique. In group 1, collagen type IV, fibronectin and laminin were absent from the luminal epithelium but present in stromal cells and the basement membrane of glands and blood vessels. In group 2, these components were absent from all endometrial regions using equivalent titres of antibody to those used in group 1. This suggests that the endometrium of women with unexplained infertility demonstrates defects in the distribution of certain ECM glycoproteins. A possible consequence of this defect may be implantation failure.     

Expression of type IV collagen in the developing human kidney

The distribution of alpha1-6 chains of type IV collagen (alpha1-6(IV)) in human fetal kidneys was examined by indirect immunofluorescence. By 11 weeks of gestation, alpha1, 2, 3, 4, and 6(IV) were already present, but alpha5(IV) appeared relatively late, at 21 weeks. Alpha1(IV) and alpha2(IV) were present in all basement membranes, alpha3(IV) and alpha4(IV) were restricted to the glomerular basement membrane and parts of the tubular basement membrane. Alpha5(IV) was distributed in the glomerular basement membrane, Bowman's capsule, and parts of the tubular basement membrane. Alpha6(IV) was present in the Bowman's capsule, parts of the tubular basement membrane, and occurred in parts of the glomerular basement membrane at the early capillary loop stage, but disappeared during the later capillary loop stage.     

Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro

Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis.     

Laminin interactions important for basement membrane assembly are promoted by zinc and implicate laminin zinc finger-like sequences

Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn2+ can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin gamma chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, and Zn2+) on laminin binding activity was evaluated. Zn2+ was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn2+ was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca2+-dependent laminin polymerization was unaffected by Zn2+, an observation consistent with the lack of zinc finger-containing repeats in the terminal globular domains required for polymerization. We conclude that Zn2+-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV.     

Afferent and efferent nerve terminal degeneration in the guinea-pig cochlea following atoxyl administration

Atoxyl causes destruction of both afferent and efferent nerve endings. Degeneration of afferent nerve terminals occurred even though the adjacent hair cell had a normal ultrastructure. The degeneration of the efferent nerve endings took place at the same time as the adjacent cell disintegration. Earlier studies on the effects of atoxyl have shown that it also induces damage to the stria vascularis and Reissner's membrane, thus interfering with endolymph metabolism (Anniko & Wers?ll, 1975; Anniko, 1975a, b). The afferent nerve terminals may be more sensitive to changes in the environment (endolymph) than are the surrounding structures, including efferent nerve endings, hair cells and supporting structures, and would therefore be the first structures to disintegrate.