Effects of antimicrotubular agents on the secretion of collagen. A biochemical and morphological study.

Embryonic chick cranial bone was cultured in the presence of the antimicrotubular agents, colchicine and vinblastine, and with a number of other compounds known from previous studies to affect the cellular handling of collagen. Secretion of procollagen, quantitated by light microscope autoradiography, was correlated with the extent of conversion of procollagen to collagen and with rates of collagen and noncollagen-protein synthesis. Colchicine inhibited procollagen secretion and conversion to collagen and specifically inhibited collagen synthesis. Cells exposed to colchicine revealed an increased number of dilated Golgi-associated vacuoles and vesicles, some of which contained parallel aggregates of filamentous structures. These observations suggest that the pathway of at least a fraction of procollagen secretion by osteoblasts includes the Golgi complex. Disruption of microtubules may interfere with the movement of Golgi-derived vesicles, and the resulting accumulation of collagen precursors in the Golgi complex may lead secondarily to an inhibition of synthesis. Although vinblastine also inhibited both procollagen secretion and conversion to collagen, the observed reduction in general protein synthesis and striking changes in the ultrastructure of the rough endoplasmic reticulum complicated interpretation of the effects. Interpretation of the effects of cytochalasin B was limited by the finding that the cellular response in cranial bone was markedly heterogeneous and that, contrary to some previous reports, the drug caused an inhibition in the incorporation of radiolabeled amino acids into both collagen and noncollagen protein.

Procollagen: conversion of the precursor to collagen by a neutral protease.

An enzymatic activity (procollagen peptidase), capable of converting the biosynthetic precursor procollagen to collagen at neutral pH, has been identified in rat and chick calvarial bone. Limited proteolysis of procollagen with chymotrypsin resulted in a similar transformation. The activity in bone can be demonstrated in vitro despite inhibition of new collagen synthesis by cycloheximide. Preservation of the collagen precursor in preparations extracted with acetic acid results from inhibition of the enzymatic activity at low pH.

Frequency dependence of electric field modulation of fibroblast protein synthesis.

The effect of electric current on protein biosynthesis in mammalian fibroblasts was investigated with neonatal bovine fibroblast-populated collagen matrices. The field strength dependence of electric field modulation of proline incorporation into extracellular and intracellular protein was measured over a frequency range from 0.1 to 1000 hertz. A frequency- and amplitude-dependent reduction in the rate of incorporation was observed. In tissues containing cells aligned either parallel or perpendicular to the electric field, this response was dependent on the orientation of the cells relative to the direction of the applied electric field. This study demonstrates that currents of physiological strength can stimulate alterations in biosynthesis and thereby may influence tissue growth, remodeling, and repair.

Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness.

Living skin-equivalent grafts consisting of fibroblasts cast in collagen lattices and seeded with epidermal cells were successfully grafted onto the donors of the cells. The grafts were vascularized, did not evoke a homograft reaction, inhibited wound contraction, filled the wound space, and persisted.

Gap junctional intercellular communication contributes to the contraction of rat osteoblast populated collagen lattices.

The contraction of native collagen lattices by resident mesenchymal cells mimics the organization of collagen during development and repair. Lattice contraction is cell density dependent, suggesting that cell-to-cell communications may contribute to the process. This possibility was investigated by comparing lattice contraction by four rat osteoblastic cell lines: ROS 17/2.8 cells (ROS); ROS transfected with an antisense cDNA sequence of the gap junctional protein connexin 43 (RCx16); ROS transfected with connexin 45 cDNA, a connexin not normally expressed in ROS cells (ROS/Cx45); and ROS transfected with cDNA encoding carboxy-terminal truncated Cx45 (ROS/Cx45tr). The cell coupling indices, which reflect gap junctional communication, were quantitated by the fluorescent dye scrape loading. ROS cells were well coupled (index 3.0), ROS/Cx45tr were better coupled (index 4.2), ROS/Cx45 were poorly coupled (index 1.7), and RCx16 showed no coupling (index 1.1). As determined by immunoblotting, the level of connexin 43 protein was increased in both ROS/Cx45tr and ROS/Cx45 cell lines compared with ROS cells, while the level in RCx16 cells was reduced. ROS populated collagen lattices (PCLs) contracted significantly more at day 5 (177 mm2 to 67 mm2) than ROS/Cx45tr (84 mm2), ROS/Cx45 (108 mm2), or RCx16 (114 mm2). Myosin ATPase activity, which is required for lattice contraction, was equivalent in all four cell lines, indicating that it was not responsible for inhibiting PCL contraction. ROS cells in collagen appeared elongated compared with the other cell lines which were more rounded. These experiments suggest gap junctional communication contributes to PCL contraction by resident osteoblasts.

Effects of increased concentrations of prostaglandin E levels with epidermolysis bullosa dystrophica recessive fibroblasts within a populated collagen lattice.

Tissue-cultured fibroblasts suspended in a collagen matrix actively reduce the size of that matrix by the process called lattice contraction. Cultured human fibroblasts derived from patients with epidermolysis bullosa dystrophica recessive (EBdr) cannot elongate and spread out when incorporated in a collagen matrix and they are therefore poor at contracting that collagen lattice. Culture medium from EBdr fibroblast-populated collagen lattice (FPCL) shows an increased concentration of prostaglandin, PGE2, compared with that in lattices made with equal numbers of normal human fibroblasts. The addition of the nonsteroid anti-inflammatory drug, indomethacin, to EBdr FPCL inhibits PGE synthesis, and promotes both cell elongation and spreading, as well as lattice contraction. However, the addition of indomethacin to normal FPCL does not stimulate either the spreading and elongation of cells or lattice contraction. PGE1 or PGE2 added to normal FPCL inhibits lattice contraction and cell elongation and spreading. Accordingly, EBdr FPCL does not undergo contraction due to altered elongation and spreading of fibroblasts, which process is related to enhanced PGE synthesis. It is proposed then that the contractile forces responsible for lattice contraction are identical to those responsible for the spreading and elongation of cells. Characteristic of EBdr fibroblasts are elevated levels of PGE2 which result in the failure of cells to spread and elongate within a collagen matrix. PGE2-treated normal cells do not readily spread and elongate and they do not readily contract FPCL.

Epidermolysis bullosa dystrophica recessive fibroblasts produce increased concentrations of cAMP within a collagen matrix.

Human dermal fibroblasts grown in tissue culture can be suspended and cultured in collagen lattices. These fibroblast-populated collagen lattices (FPCL) undergo a reduction in size by the process of lattice contraction. Fibroblasts from patients with epidermolysis bullosa dystrophica recessive, EBdr, produce excessive quantities of cAMP. These high concentrations of cAMP may be related to the inability of the EBdr fibroblast to elongate and spread out when incorporated into the collagen matrix. Fibroblasts with these morphologic characteristics are not effective in contracting collagen lattices. EBdr fibroblasts in FPCL have intracellular concentrations of cAMP 8 times greater than those of normal fibroblasts in FPCL. They also have a dendritic morphology. The addition of cholera toxin or dibutyryl cAMP to normal human fibroblasts will cause elevated levels of intracellular cAMP and will inhibit the elongation and spreading of cells and lattice contraction. The cytoskeletal morphology of EBdr fibroblasts differs from that of normal human fibroblasts in FPCL. The use of rhodamine phalloidin, a specific fluorescent stain for F-actin filaments, reveals that EBdr fibroblasts show a pattern of actin distribution shared by normal fibroblasts cultured in the presence of dibutyryl cAMP or cholera toxin. It is proposed that the contractile forces responsible for lattice contraction are identical to those forces responsible for the spreading and elongation of cells. EBdr fibroblasts fail to spread and elongate within a collagen matrix and are therefore not effective in lattice contraction.

ATP-induced cell contraction with epidermolysis bullosa dystrophica recessive and normal dermal fibroblasts.

Human dermal fibroblasts cultured on glass coverslips and permeabilized by glycerol can be induced to undergo cell shrinkage by the addition of ATP in buffer containing calcium and magnesium. They reduce in size by 72% in 10 min. ATP-induced cell contraction is linked to an aggregation of cytoplasmic filaments as demonstrated by rhodamine-phalloidin F-actin staining. Before the addition of ATP, glycerinated cells have parallel arrangements of staining cytoplasmic filaments. Afterward, dermal fibroblasts from patients with epidermolysis bullosa dystrophica recessive (EBdr) show only a 10-20% reduction in cell size, and little F-actin aggregation staining can be demonstrated. Epidermolysis bullosa dystrophica recessive fibroblasts have been reported to produce excess prostaglandin E2 (PGE2) and cAMP. The preincubation of normal dermal fibroblasts for 24-30 h with 10 micrograms/ml PGE2, 10 micrograms/ml cholera toxin, or 1 mM dibutyl cAMP will reduce ATP-induced cell contraction to less than 20%. Treated cells showed little disruption of cytoplasmic F-actin. Epidermolysis bullosa dystrophica recessive fibroblasts preincubated with the cyclooxygenase inhibitor indomethacin at 10 micrograms/ml restored cell contraction to 74%. These treated cells also show aggregation of F-actin filaments. The process of ATP-induced cell contraction can be altered by the intracellular concentrations of cAMP, the levels of which are elevated in the fibroblasts in EBdr patients. A mechanism for cAMP inhibition of cell contraction is discussed.

Epidermolysis bullosa dystrophica recessive fibroblasts altered behavior within a collagen matrix.

Normal human fibroblasts incorporated into a collagen lattice reduce the size of that lattice over a period of time. Lattice size reduction or lattice contraction is directly related to initial cell number. When equal numbers of fibroblasts derived from patients with epidermolysis bullosa dystrophica recessive, (EBdr), are used, there is delayed lattice contraction. The EBdr fibroblasts have an altered cellular shape, when compared to normal cells, in that the EBdr cells fail to flatten out and elongate, but do attach to collagen fibers like normal fibroblasts. EBdr fibroblasts maintain a rounded shape with numerous filopodia radiating from the cell periphery and such filopodia are attached to the collagen fibers of the lattice. In monolayer tissue culture on glass surfaces, EBdr fibroblasts are three times more likely to grow over neighboring fibroblasts. EBdr cell filopodia structures are attached to the cell surfaces lying beneath them, which demonstrates another condition of altered anchorage attachment of EBdr fibroblasts.

Dexamethasone abrogates the fibrogenic effect of transforming growth factor-beta in rat granuloma and granulation tissue fibroblasts.

Administration of TGF-beta, a fibrogenic inflammatory growth factor, promotes fibrosis and scarring. Dexamethasone, an anti-inflammatory steroid, inhibits wound healing and reduces fibrosis. The current studies were initiated to determine whether the co-administration of dexamethasone was able to abrogate the fibrogenic effect of TGF-beta. Polyvinyl alcohol sponges were implanted subcutaneously on the abdominal area of rats and directly injected with vehicle, dexamethasone, TGF-beta, or dexamethasone plus TGF-beta. Dexamethasone was able to block the fibrogenic effect of TGF-beta. Collagen and noncollagen protein synthesis was measured as a function of TGF-beta or dexamethasone concentrations in fibroblasts isolated from granulation tissue. Addition of dexamethasone to cultures treated simultaneously with TGF-beta blocked the fibrogenic response of TGF-beta. To study the molecular regulation of collagen gene expression by TGF-beta or dexamethasone, fibroblasts derived from granulation tissue were stably transfected with the ColCat 3.6 plasmid, which contains the rat pro alpha1(I) collagen promoter linked to the chloramphenicol acetyltransferase (CAT) gene. Dexamethasone decreased CAT activity whereas TGF-beta increased the activity of this reporter gene. The increase in CAT activity observed with TGF-beta treatment was significantly decreased when dexamethasone was added to the cultures, although CAT activity did not return to control level. Since collagen synthesis in fibroblasts treated simultaneously with dexamethasone and TGF-beta1 was found to be the same as that of untreated samples, the data indicate that there is a dexamethasone-mediated posttranscriptional regulation of pro alpha1(I) collagen mRNA. These studies demonstrate that at the in vivo level, the cellular level, and the molecular level, dexamethasone is able to block the fibrogenic effect of TGF-beta.

Promotion of vascular patency in dermal burns with ibuprofen.

Differences in the vascular response to burn and freeze injuries were investigated as a model for defining the mechanism and cause of vascular occlusion in rats after dermal burns. Concentrations of thromboxane and prostacyclin in wound fluid were elevated in both types of trauma. However, inhibition of prostaglandin synthesis by indomethacin failed to promote vascular patency in burn-injured animals. However, the systemic administration of ibuprofen and imidazole led to increased vascular patency. Ibuprofen promoted vascular patency even when given six hours after burn trauma. These studies indicate that ibuprofen and imidazole promote vascular patency by fostering fibrinolysis rather than by inhibiting prostaglandin synthesis and release.

Regenerative healing of incisional wounds in midgestational murine hearts in organ culture.

OBJECTIVE: Using an organ-culture fetal heart repair model, we explored fetal repair in tissues other than dermis. METHODS: Wounded fetal mouse hearts of 14 and 18 days' gestation (term = 20 days), as well as hearts of 22 days' gestation (newborn), were maintained in serum-free medium. Specimens were fixed at 2, 7, and 11 days and then processed for histologic examination. Small fragments of fetal hearts from all time points were cultured as explants. The migration of cells from the periphery of the explants was compared at day 4, and the pattern of microfilaments in these cells was assessed. RESULTS: In 14-day hearts (n = 18), tissue architecture was rapidly reestablished without an inflammatory response or scarring, constituting regenerative repair. In 18-day hearts (n = 18), no reestablishment of muscle fibers or wound closure occurred. In the 22-day explants (n = 12) the wounds closed by scarring. Cell migration from 14-day explants was 4.7 +/- 2.3 ocular units; from 18-day explants, it was 2.6 +/- 1.1 ocular units; and from 22-day explants, it was 0.9 +/- 0.4 ocular units. Microfilaments of 14-day cells were arranged at the periphery of the cell consistent with cardiomyocytes. Microfilaments of 18- and 22-day cells were arranged in parallel arrays (stress fibers) that were consistent with fibroblasts. CONCLUSIONS: We propose that regenerative healing of 14-day fetal hearts is by the migration of cardiomyocytes. At 18 and 22 days, cardiomyocytes are too differentiated and unable to migrate; hence cell migration is limited to resident fibroblasts, which are deficient at 18 days but sufficient at 21 days to be repaired by the scarring process.

Vanadate and the absence of myofibroblasts in wound contraction.

HYPOTHESIS: Fibroblasts, not myofibroblasts, are responsible for wound contraction. Only myofibroblasts express a smooth muscle actin for which vanadate blocks its expression. Wound contraction in vanadate-treated rats will proceed normally in the absence of myofibroblasts. DESIGN: Laboratory study using rats. METHODS: Wound healing in rats receiving vanadate parenterally, an inhibitor of tyrosine phosphate phosphatases, was investigated. For 21 days, treated rats received drinking water containing vanadate, 0.2 mg/mL, in isotonic sodium chloride solution, and the control rats received isotonic sodium chloride solution alone. On day 7, 4 square, full-excision wounds were made dorsally and measured, then 2 polyvinyl alcohol sponges were placed ventrally in subcutaneous pockets. RESULTS: After 2 weeks, the wound area in the rats receiving vanadate measured 7.1 +/- 1.8 U (mean+/-SD), and the wound area in the control rats measured 7.2 +/- 2.2 U. The control rats' granulation tissue (GT) had myofibroblasts, or alpha-smooth muscle (alpha-SM) actin-positive fibroblasts, whereas the vanadate-treated group's fibroblasts were devoid of alpha-SM actin. By Western blot analysis, GT homogenates in the vanadate-treated group contained less alpha-SM actin. By electron microscopy, control rats' GT showed classic myofibroblast populations, and the collagen fiber bundles were randomly organized. In contrast, the wounds in the vanadate-treated group showed unencumbered fibroblast populations and neatly ordered, parallel collagen fiber bundles. By polarized light microscopy, the GT of the vanadate-treated group displayed orderly collagen fiber bundles. CONCLUSIONS: The differentiation of fibroblasts into myofibroblasts requires the dephosphorylation of selected tyrosine phosphate residues. In the absence of myofibroblasts, the rate of rat wound contraction is normal, and collagen fiber bundles have a more orderly arrangement. Myofibroblasts are not required for wound contraction.

Ibuprofen as an antagonist of inhibitors of fibrinolysis in wound fluid.

Fibrin plate assay revealed that rat serum and wound fluid harvested from seven day subcutaneously implanted wound chambers prevented fibrinolysis. Samples of wound fluid from one to four hour burns displayed greater inhibiting activity than unburned or 24 hour old burns. Ibuprofen, a non-steroid anti-inflammatory drug, reversed the blocking of fibrinolysis in wound fluid, but it had no action on rat serum. The activity of ibuprofen appears unrelated to the synthesis of prostanoids. Fractionation of wound fluid and serum by column chromatography showed differences in elutions of inhibitors of fibrinolysis. Serum fractions having molecular weights greater than 60,000 prevented fibrinolysis and they were unaffected by the addition of ibuprofen. Fractionations of wound fluid produced a number of inhibitors, some of which had molecular weights of approximately 40,000. This inhibitor(s) was not detected in serum and was reversed by adding ibuprofen. Wound fluid has a fibrinolytic inhibitor which differs from that in the circulatory system, and which may be critical to the vascular changes of burn trauma.

Evidence for the involvement of microtubules in wound contraction.

Many theories have been proposed for the mechanism of wound contraction, that phenomenon of wound closure in which the skin surrounding the tissue defect is drawn into the open wound. When agents that inhibit microtubular function, such as vinblastine and colchicine, were topically applied to actively contracting wounds, contraction stopped. Cytochalasin B, an agent that reportedly disrupts microfilaments, did not alter contraction. These results suggest that wound contraction is related to the functioning of microtubules in fibroblasts within the wound and is proceeding at its maximal rate. The results tend not to support the theory that the microfilament components of cells are involved in wound contraction.

The modulation of contraction of fibroblast populated collagen lattices by types I, II, and III collagen.

Human dermal fibroblasts incorporated in a polymerized collagen lattice reduce the size of that matrix. When cell number, collagen concentration, and medium are identical, lattices made with type III collagen contract faster and to a greater degree than those made with type I collagen. The latter contract faster and to a greater degree than those made with type II collagen.

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction.

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.

Differences in cell division and thymidine incorporation with rat and primate fibroblasts in collagen lattices.

Human and gorilla dermal fibroblasts, primate cells, suspended in a collagen lattice, do not divide for the first 3 days. In contrast, rat fibroblasts divide within 24 hr. In this study, the proliferation of rat fibroblasts were compared to primate fibroblasts. Rat fibroblasts in monolayer culture increase from 100,000 to 355,000 in 2 days, and human cells increase from 100,000 to 436,000 in the same period. An initial seeding of 100,000 rat fibroblasts suspended in collagen increased to 163,000 cells in 2 days. An initial 100,000 human fibroblasts seeded in collagen decreased to 80,000 cells in 2 days. Retarded proliferation of human and gorilla fibroblasts in collagen is unrelated to a defect in DNA synthesis. By autoradiography human fibroblasts suspended in collagen incorporate labelled thymidine. By flow cytometry analysis, the DNA concentrations of human fibroblasts suspended in collagen exhibited 41% in a 4N chromosome state, compared to 14% in monolayer culture. Nuclei of gorilla fibroblasts from collagen displayed 42% in a 4N state, compared to 19% in monolayer culture. With nuclei of rat fibroblasts from collagen, 14% were in a 4N state, compared to 9% in monolayer culture. Primate fibroblasts show a three-fold increase in the number of nuclei in a 4N state compared to rat fibroblasts suspended in collagen. After replating fibroblasts released from collagen in monolayer culture in the presence of 1 mM hydroxyurea (an inhibitor of DNA synthesis) primate fibroblasts doubled in 24 hr. Under identical conditions, rat fibroblasts showed no cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Free fatty acids and dialyzed serum alterations of fibroblast populated collagen lattice contraction.

Fibroblast populated collagen lattices (FPCL) have facilitated the in vitro study of wound contraction and scar contracture. Mixing fibroblasts, serum containing culture medium and soluble collagen, together and then incubating the mixture at 37 degrees C produces a FPCL. The fibroblasts elongate and spread within the collagen matrix, and by forces associated with cell locomotion they reorganize the collagen fibers. The reorganization of the collagen produces a reduction in size of the FPCL, called lattice contraction. It was also found that dialyzed fetal bovine serum did not support lattice contraction. Supplementing dialyzed serum with fatty acids accelerated lattice contraction. The fatty acid composition of the fibroblast plasma membrane influences that membrane fluidity. These studies demonstrated that lattice contraction was enhanced by the additions of saturated fatty acids in the order of laurate (C-12), palmitic (C-16), and stearate (C-18). With unsaturated fatty acids additions, the order of enhanced lattice contraction was arachidonate (4 C = C), linoleate (2 C = C) and oleate (1 C = C). The addition of dialyzed serum with or without fatty acids neither altered ATP-induced cell contraction activity nor cell proliferation. It was concluded that free fatty acid additions do not modulate FPCL contraction by enhancing microfilaments contraction or increasing cell numbers. The mechanism of action was proposed to be by altering cell membrane fluidity. This finding further supports the theory that the mechanism for lattice contraction is cell locomotion, rather than cell contraction.

In vitro ligation of ureters and urethra modulates fetal mouse bladder explants development.

The compliance of the bladder which accommodates the holding and voiding of urine is influenced by the amount and type of collagen deposited as well as the packing and organization of collagen fiber bundles. During fetal development, the accumulation of urine within the bladder lumen is associated with the maturation of the bladder's wall. Fetal mouse bladders can undergo maturation as organ cultured explants in defined medium. Polarized light optics of Sirius red-stained sections of fetal mouse bladders in organ culture for 4 days showed that the ligation of both ureters and urethra promoted more orderly packing of collagen fiber bundles within the luminal edge of the lamina propria compared to unligated bladder explants. It is proposed that ligation causes differences in the development and organization of the collagen fiber bundles within the bladder wall. These differences are due to either increases in intravesical pressure, the accumulation of growth factors within the lumen or a combination of both.