Eijiro Adachi

In vitro Formation of Hybrid Fibrils of Type v Collagen and Type I Collagen Limited Growth of Type I Collagen Into Thick Fibrils by Type V Collagen

Type V collagen and type I collagen were obtained from human placentas by pepsin treatment, followed by salt fractionation. The precipitates formed at 37 degrees C from a mixed solution of type V collagen and type I collagen, reacted with antibodies to either type V collagen or type I collagen. The precipitates seen by electron microscopy were fine flexible fibrils, with a D-periodic banding pattern. The average diameter of hybrid fibrils was smaller than 50 nm, when the proportion of type V collagen exceeded that of type I collagen. Type V collagen directly interacts with type I collagen in forming hybrid fibrils, resulting in limitation of the growth of type I collagen fibrils into thicker fibrils. We propose that the fibrils with a predominant type V collagen content may occur in the pericellular environment of various tissues, as a basic structure in connecting basal laminae with interstitial collagen fibrils.

Growth Rate of Human Fibroblasts is Repressed by the Culture within Reconstituted Collagen Matrix but not by the Culture on the Matrix

Effect of reconstituted type I collagen gel on in vitro growth of fibroblasts was examined. A prolonged lag period was observed by culture either on the surface of three-dimensional collagen gel (on-gel) or within the gels (in-gel) as compared with the culture on plastic dishes. The rate of cell proliferation in logarithmic phase growth was repressed by the culture in-gel but not by the culture on-gel. The differential growth rates between in-gel culture and on-gel culture should be ascribed to difference in distributions of interacting sites between cell and collagen fibrils. The repression of cell growth was more marked in a contracting collagen gel which contains higher density of collagen fibrils. The cell density in contracted gel per unit cross-section area was found to be much lower than that of confluent monolayer culture. These results suggest that the repression of cell growth by collagen fibrils in the three-dimensional gels is not due to direct cell-cell contact, but due to the distribution and number of contact sites between a cell and collagen fibrils.

A comparison of the immunofluorescent localization of collagen types I, III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata)

SummaryLocalizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens.

Dissociation of actin microfilament organization from acquisition and maintenance of elongated shape of human dermal fibroblasts in three-dimensional collagen gel

Actin microfilaments of the fibroblasts cultured in a collagen gel were distributed along the inner surface of the entire cell membrane, in either spherical shape at an initial stage of culture or elongated shape at a later stage. The distribution was quite different from that of the fibroblast cultured on a two-dimensional surface, where actin microfilaments were found to be aligned essentially along the inner membrane which is in contact with a flat surface. Timing of morphological change from spherical shape to spread shape or elongated shape was also greatly affected by contact with substrates whether in two-dimension or in three-dimension: distinct morphological change was observed within 6 h on glass or on the collagen gel, and at 30 h or later within the collagen gel. The retardation of cell elongation in the gel was antagonized by a low dose (0.2 microM) of cytochalasin D, although the drug kept the cells in round shape at a concentration of 2 microM. Since a low concentration of cytochalasin was reported to induce actin polymerization in vitro, the organization of actin microfilaments was examined by rhodamine-phalloidin staining. It was found that actin filaments in elongated cells by low cytochalasin D were disrupted. These results suggest that accelerated acquisition of elongated shape by the treatment of a low dose of cytochalasin D might be initiated by destabilization of the actin microfilaments that may scaffold the spherical shape of the cell in the collagen gel. The elongated shape thus formed returned to spherical upon washing of the added free cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopical evidence that type V collagen is a fibrillar collagen: importance for an aggregating capability of the preparation for reconstituting banding fibrils

Type V collagen has already been shown, in many immunohistochemical studies, to be widely distributed in connective tissues. Its supramolecular structure, however, has been unclear. We demonstrate that the major aggregates formed from type V collagen solution in vitro are fine fibrils with a D-periodic banding pattern. Further, by using the immunogold labeling method, we find that these fibrils react strongly with anti-type V collagen antibody. Electronmicroscopic examination showed three kinds of aggregate: fine fibrils with periodic banding pattern, fine fibrils without banding pattern, and non-fibrillar materials. Both striated and nonstriated fibrils, when incubated with rat polyclonal anti-human type V collagen IgG followed by incubation with 15 nm-gold conjugated goat anti-rat IgG, were labeled with colloidal gold. We conclude that type V can be classified as a fibrillar collagen. Also, from the present findings together with previous studies, we believe type V collagen may exist in vivo in various connective tissues as fine fibrils with a 67 nm-periodic banding pattern, by itself, or with type I or type III fibrillar collagen, being located between, and connecting the basal lamina and interstitial collagen fibers.

Optic nerve regeneration by peripheral nerve transplant

We studied the morphology of regenerated retinal ganglion cells and their axons in adult rodents after axotomy and autologous transplantation of the sciatic nerve. Regenerated ganglion cells, backlabeled with rhodamine dextran, were of similar size to or larger than those of intact cells in control animals. Dendrites and occasionally axons as well showed abnormal morphologies in most cells, though some cells appeared quite normal. Cross-sections of the regenerated axons, observed by electron microscopy, were always attached to either the Schwann cell cytoplasm or the basal lamina. The immunoreactive structures to anti-laminin antibody were quite irregular in the cross-sectioned graft and, compared with those of the intact sciatic nerve, they were generally smaller. Their appearance closely resembled that of the basal lamina in the graft observed by electron microscopy. These observations, taken together, suggest that the laminin-rich basal laminae of Schwann cells are essentially important for the regeneration of retinal axons in adult rodents.

Changes of compound action potentials in retrograde axonal degeneration of rat optic nerve

Electrophysiological recordings of compound action potentials (CAPs) were made from the optic nerve (ON) following unilateral optic tract (OT) transection in adult rats. In response to optic chiasm stimulation CAPs recorded from the ON ipsilateral to the transected OT, i.e., the ON of control side, revealed three positive waves, termed n1, n2, and n3 components, corresponding to fast-, intermediate-, and slow-conducting fibers. Two weeks after OT transection, CAPs recorded from the ON contralateral to the transected OT, i.e., the ON of cut side, were generally smaller in amplitude than those recorded from the ON of control side. The degree of amplitude reduction was different among three components; n3 component was severely reduced and n2 component was moderately reduced, whereas n1 component relatively persisted. These tendencies were more marked in CAPs recorded at 4 weeks after OT transection; n3 component disappeared, whereas n1 and n2 components decreased in amplitude with elongation of latency. Cross sections of the ON after unilateral OT transection were examined electron microscopically, in which significant decrease in fiber density, demyelination, and distorted fibers were verified in the cut side. The present study suggests that slower-conducting ON fibers, that is, smaller-diameter ON fibers are initially and remarkably thrown into retrograde degeneration, while larger-diameter ON fibers are relatively resistant to axotomy.

Comparison of axial banding patterns in fibrils of type V collagen and type I collagen.

Type V collagen and type I collagen were obtained from human placenta, essentially by salt fractionation. Precipitates were formed from mixed solutions of type V collagen and type I collagen in various ratios. They were incubated at 37 degrees C for 1 hour and negatively stained with 0.5% uranyl acetate (pH 4.4) at 37 degrees C. The specimens, seen by electron microscopy, were fibrils with a D-periodic banding pattern. Axial electron density profiles of collagen fibrils were obtained from selected electron micrographs by densitometric tracing. The slit width corresponded to 1.5 nm. The relative electron densities of overlap region vs. hole region were lower than 20% in fine fibrils containing a significant amount of type V collagen. It is suggested that the overlap region of such collagen fibrils may be loosely packed, being accessible to uranyl acetate, or the hole region may be filled by larger non-collagenous portions of type V collagen, resulting in loss of the light and dark alternation. Six to 8 white transverse lines were discerned per period and labeled consecutively with Arabic numerals. White lines 2 and 5 tended to merge with lines 1 and 4, respectively, in collagen fibrils formed from a solution containing a significant amount of type I collagen or pure type I collagen. The eight white lines corresponded to c2, c1, b2, b1, a4, a1, e1 and d with reference to their locations in the D-period. The locations of white lines in collagen fibrils which contain significant amount of type V collagen were identical with those in type I collagen fibrils. This is consistent with the primary structure that the axial distribution of charged amino acids in type V collagen is quite similar to that in type I collagen.

Type V Collagen in Splenic Reticular Fibers of the Macaque Monkey

The distribution of type I, III and V collagens in the monkey spleen was examined by indirect immunofluorescent microscopy and immunoelectron microscopy, and compared with that of reticular fibers revealed by a silver impregnation method. Type I collagen was localized on reticular fibers in the white pulps and on coarse reticular fibers in the splenic cords. Type III collagen was localized on the reticular fibers in the white pulps, and on the coarse reticular fibers and a limited number of fine reticular fibers, in the splenic cords. The anti-type V collagen antibody reacted with annular reticular fibers around the splenic sinuses, as well as with the reticular fibers in the white pulps and with the coarse and fine reticular fibers in the splenic cords. Thus, the distribution pattern of fibers that reacted with the anti-type V collagen antibody was very similar to that of the reticular fibers revealed by the silver impregnation method. Electron-microscopically, the fine reticular fibers in the splenic cords were composed of collagen fibrils, 30-50 nm in diameter, and amorphous substances. They were covered by reticular cell processes. By immunoperoxidase labeling with the anti-type V collagen antibody, electron-dense reaction products were found over the collagen fibrils with a banding pattern. These results indicate that type V collagen is an indispensable component of the reticular fibers.

Alteration of collagen IV in acutely deteriorated renal allografts.

BACKGROUNDnThe changes in the basement membrane occurring in acutely deteriorated renal allografts (ADR) have not been extensively investigated. Our purpose is to elucidate the alteration of collagen IV, a main constituent of the basement membrane in ADR.nnnMETHODSnFifty biopsy specimens of ADR and 10 of chronic transplant nephropathy (CTN) were examined with two monoclonal antibodies specific for collagen IV. JK199 and JK132 are monoclonal antibodies that recognize triple helical collagen IV containing the alpha1 chain. JK199 recognizes all the basement membrane containing [alpha1 (IV)]2alpha2(IV), although JK132 reacts only with a limited portion of it. In the normal kidney, JK199 reacts with the mesangial matrix, the basement membrane of Bowmans capsule (BBM), and the tubular basement membrane, as well as with the glomelular basement membrane (GBM). JK132 reacts with the mesangial matrix, BBM, and the tubular basement membrane.nnnRESULTSnIn ADR, increased intensity of JK199 was observed in GBM, the mesangial matrix, BBM, the tubular basement membrane, and the interstitium. Increased intensity of JK132 was observed in the mesangial matrix, BBM, and the tubular basement membrane, but was not remarkable in GBM or the interstitium. In contrast, biopsy specimens of CTN showed increased intensity of JK132 in GBM, the mesangial matrix, BBM, the tubular basement membrane and the interstitium.nnnCONCLUSIONnThese results suggest that collagen IV is up-regulated in ADR. Differential staining of collagen IV with JK199 and JK132 in GBM and the interstitium may contribute to diagnose CTN.