Structural modulations affecting a small fraction of the population of plasmalemmal vesicles of vascular endothelia are described. They include forms which are apparently produced by the fusion of the vesicular membrane with the plasmalemma and by the successive elimination of the layers of the two fused membranes. Such modulations are assumed to represent stages in the discharge process of vesicular contents. Other forms, characterized by their flask shape and elongated neck, are assumed to represent stages in the formation and loading of membrane invaginations, followed by their being pinched off to form isolated vesicles. Stages in a membrane-fusion process leading to the formation of apertured fenestrae and channels are also described in fenestrated endothelia. The visualization of these structural details is greatly facilitated by staining tissue specimens with uranyl acetate before dehydration.According to a previously advanced hypothesis (1-4), transport of fluid in quanta across the endothelium of blood capillaries implies the following sequence of events: (a) appearance of a pocket by invagination of the plasmalemma on either cell front; (b) formation of a closed vesicle by a pinching off (membrane fission) of the pocket; (c) movement of the vesicle across the endothelial cytoplasm; (d) fusion of the vesicle membrane with the plasmalemma on the opposite cell front; and finally (e) discharge of the vesicular content into the extracellular medium.Most electron micrographs so far published seem to represent either (1) invaginations of the cell membrane or (2) closed vesicles assumed to be in transit across the endothelial cell. Recently, intermediate stages have tentatively been identified in the form of vesicles provided with a narrow neck (3) or an aperture (diaphragm) (5, 6). The first are considered intermediary forms between (a) and (b), whereas the second are assumed to represent vesicles approaching the discharge stage (3, 5, 6).A priori, clear visualization of the stratified structure of plasmalemmal and vesicular membranes should allow the recognition of more graded intermediary forms, especially during those phases of the process which involve membrane fusion prior to vesicle opening and discharge. For instance, appearances representing contact and fusion of the two membranes, as well as subsequent progressive elimination of their layers, should be encountered with a frequency proportional to the time taken by these events in a complete traverse of the endothelium.The present paper reports the occurrence of modulations in vesicle structure which probably represent such intermediate stages. MATERIALS AND METHODSOur observations were carried out on (a) blood capillaries of the tongue, myocardium, diaphragm, intestine, and pancreas; (b) lymphatic capillaries of the tongue and intestine; and (c) endocardium of adult rats and guinea pigs.

Abstract. Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res.,. Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive : identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat antimouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and IU labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat taft tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen. WITH the discovery of the genetic heterogeneity of collagen (38), it became apparent that the composition and organization of the extracellular matrices were far more complex than were ever anticipated. To date there are at least 10 known, distinct collagen gene products, not including non-matrix collagen sequences such as those in Clq (47, 48), acetylcholine esterase (50), and surfactant (60). The molecular structure and tissue supramolecular aggregates of most of these are established. For several, however, the forms in which they exist in situ and their relations with other matrix components are not yet known. Type VI collagen is an example of the latter.Extracellular filaments and fibrils that have a 100-nm periodic cross-banding have been observed in chicken embryo fibroblast cultures (7). The basic unit, a "beaded" filament, consists of a thread measuring 2-3 nm in diameter and pairs of "beads" distributed along its length at regular intervals of 90-110 nm. In the aggregated form (i.e., bundles of ordered "beaded" filaments), the...

The wall of the blood capillaries of skeletal muscles (diaphragm, tongue, hind legs) and myocardium of the rat, guinea pig, and hamster consists of three consecutive layers or tunics: the endothelium (inner layer), the basement membrane with its associated pericytes (middle layer), and the adventitia (outer layer). The flattened cells of the endothelium have a characteristic, large population of cytoplasmic vesicles which, within the attenuated periphery of the cells, may attain a maximum frequency of 1 20 /M 2 of cell front and occupy -18% of the cytoplasmic volume; these values decrease as the cells thicken toward the perikaryon. The vesicles are 650-750 A in over-all diameter and are bounded by typical unit membranes. They occur as single units or are fused to form short chains of two to three vesicles. Each configuration may lie entirely within the cytoplasm or open onto the cell surface. In the latter case, the unit membrane of the vesicle is continuous, layer by layer, with the plasmalemma. Chains of vesicles opening simultaneously on both the blood and tissue fronts of the endothelial tunic have not been observed either in sections or in a tridimensional reconstruction of a sector of endothelial cell cytoplasm. Adjacent endothelial cells are closely apposed to one another and appear to be joined over a large part of their margins, possibly over their entire perimeter, by narrow belts of membrane fusion (zonulae occludentes). Except for tongue capillaries, patent intercellular gaps are rare or absent. The middle layer is formed by a continuous basement membrane (500 A thick) and by pericytes which lie in between leaflets of this membrane. The tips of the pericyte pseudopodia penetrate through the inner leaflet of the basement membrane and join the endothelium in maculae occludentes. The adventitia is a discontinuous layer comprising cellular (macrophages, fibroblasts, mast cells) and extracellular (fibrils, amorphous matrix) elements. The same general type of construction appears to be used along the entire length of the capillary.

The pathway by which intravenously injected ferritin molecules move from the blood plasma across the capillary wall has been investigated in the muscle of the rat diaphragm. At 2 min after administration, the ferritin molecules are evenly distributed in high concentration in the blood plasma of capillaries and occur within vesicles along the blood front of the endothelium. At the 10-min time point, a small number of molecules appear in the adventitia, and by 60 min they are relatively numerous in the adventitia and in phagocytic vesicles and vacuoles of adventitial macrophages. Thereafter, the amount of ferritin in the adventitia and pericapillary regions gradually increases so that at 1 day the concentration in the extracellular spaces approaches that in the blood plasma. Macrophages and, to a lesser extent, fibroblasts contain large amounts of ferritin. 4 days after administration, ferritin appears to be cleared from the blood and from the capillary walls, but it still persists in the adventitial macrophages and fibroblasts. At all time points examined, ferritin molecules within the endothelial tunic were restricted to vesicles or to occasional multivesicular or dense bodies; they were not found in intercellular junctions or within the cytoplasmic matrix. Ferritin molecules did not accumulate within or against the basement membranes. Over the time period studied, the concentration of ferritin in the blood decreased, first rapidly, then slowly, in two apparently exponential phases. Liver and spleen removed large amounts of ferritin from the blocd. Diaphragms fixed at time points from 10 min to 1 day, stained for iron by the Prussian Blue method, and prepared as cleared whole mounts, showed a progressive and even accumulation of ferritin in adventitial macrophages along the entire capillary network. These findings indicate: (1) that endothelial cell vesicles are the structural equivalent of the large pore system postulated in the pore theory of capillary permeability; (2) that the basement membrane is not a structural restraint in the movement of ferritin molecules across the capillary wall; (3) that transport of ferritin occurs uniformly along the entire length of the capillary; and (4) that the adventitial macrophages monitor the capillary filtrate and partially clear it of the tracer.

This report explores the mechanism of spontaneous closure of full-thickness skin wounds. The domestic pig, often used as a human analogue for skin wound repair studies, closes these wounds with kinetics similar to those in the guinea pig (mobile skin), even though the porcine dermis on the back is thick and nearly immobile. In the domestic pig, as in the guinea pig, daily full-thickness excisions of the central granulation tissue up to but not including the wound edges in both back and flank wounds do not alter the rate or completeness ofwound closure or the final pattern of the scar. A purse-string mechanism of closure was precluded by showing that surgical interruption of wound edge continuity does not alter closure kinetics or wound shape. We conclude that "tightness" of skin is not a key factor nor is the central granulation tissue required for normal wound closure. These data imply that in vitro models such as contraction of isolated granulation tissue or of the cell-populated collagen lattice may not be relevant for understanding the cell biology of in vivo wound closure. Implications for the mechanism for wound closure are discussed.Cellular mechanisms whereby open full-thickness excision wounds in adult mammalian skin are closed spontaneously are speculative (1-6). The currently prevailing hypothesis (6, 7), originally stated in 1956 (8,9), proposes that the central granulation tissue generated shortly after wounding is a contractile machine that, through an undefined action of its fibroblasts, pulls the edges of the wound together. Recent papers on the subject (4, 7, 10, 11) promulgate the idea that a significant fraction of the mesenchymal cells of the granulation tissues, called myofibroblasts (12), have contractile powers that are exerted on collagen fibers, other matrix components, and each other. Observations by Harris and colleagues (13,14) on the mechanical effects of fibroblast traction on the organization of fibrous collagen lattices led Ehrlich (15, 16) to an alternative hypothesis that proposes that "cells (fibroblasts) working as single units use cell locomotion forces to reorient the collagen fibrils associated with them" (15). The implication is that the reoriented matrix collagen of the granulation tissue transmits the contractile force (4). However, wounds close at a normal rate in scorbutic animals (8,17) in which new collagen production is blocked, thus posing a serious hurdle for this concept unless one considers that other matrix components such as fibronectin (11,18) have the necessary tensile properties.The mechanism proposed by Abercrombie and colleagues (8, 9) was reexamined by Grillo and associates in 1958 (19) by using square full-thickness excision wounds in the guinea pig skin. Biochemical analyses of wound contents led the authors to question the proposed role of the central granulation tissues. Total removal of granulation tissues up to but not including the The publication costs of this article were defrayed in part by page charge payment. This article must theref...