Gerald M. Fuller

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.

Rotenone inhibition of spindle microtubule assembly in mammalian cells.

Abstract Rotenone, a potent inhibitor of mitochondrial respiration is also an effective antimitotic agent. The addition of either rotenone or Colcemid to exponentially growing Chinese hamster ovary cells resulted in a dramatic increase in mitotic index after 90 min. When the cultures were washed free of the drugs, mitosis was completed and the cells progressed into G 1 at approximately the same rate. Further similarity of rotenone-arrested cells to Colcemid-induced mitotic inhibition was apparent at the ultrastructural level. Mitotic cells treated by either drug contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center. Occasional microtubules were seen near the kinetochore and centrioles. These observations, along with the fact that rotenone inhibited the binding of 3H-colchicine to isolated bovine brain tubulin, suggested that rotenone inhibited mitosis by binding directly to tubulin to prevent microtubule assembly.

The effects of leucocytic and serum factors on fibrinogen biosynthesis in cultured hepatocytes.

Abstract Primary monolayer cultures of fetal rat liver cells have been used to determine whether factors derived from sera and/or leucocytes from traumatized animals can act directly on hepatocytes to cause an increased synthesis of fibrinogen, an acute-phase reactant. The data show that fibrinogen synthesis increases in a dose-response relationship by the addition of leucocytic supernatant preparations from cells of rabbits that had previously been subjected to a peritoneal injection of sterile glycogen. The response of the hepatocytes to leucocytic factor is independent of the presence of fetal bovine serum. In other experiments, fibrinogen synthesis was also significantly stimulated over controls when hepatocytes were incubated in medium supplemented with dialyzed sera from either turpentine-stimulated or sham-operated rats. These findings lend support to the notion that there is a non-dialyzable factor(s) in the sera of traumatized animals that interacts with hepatocytes to elicit the acute-phase response.

Two simple programs for the analysis of data from enzyme-linked immunosorbent (ELISA) assays on a programmable desk-top calculator

Abstract We have designed two programs for use with an inexpensive programmable calculator which rapidly and accurately convert raw data generated from enzyme-linked immunosorbent assays directly into antigen concentration. The first program computes and compares effective doses (ED50)s between a standard and each unknown sample assayed. The ED50 from the unknown sample is then multiplied by a concentration factor which yields the unknown concentration. The second program linearizes the sigmoidal enzyme-linked immunosorbent assay titration curve using a logit-log transformation of the data in order to compute unknown concentration values. Both programs employ stringent limit conditions to decrease “nonsense” calculations. Data are then processed by a least-squares best-fit linear regression analysis.

Is melatonin a microtubule inhibitor

Abstract The effect of melatonin (5-methoxy- N -acetyltryptamine) on microtubule assembly was assessed by means of viscometry, cell kinetics and [ 3 H]colchicine binding studies. Evidence presented shows that melatonin has no effect on the in vitro assembly of bovine brain microtubules. [ 3 H]Colchicine binding is not inhibited by melatonin in either crude or purified tubulin preparations. Furthermore, no increase in mitotic index is observed when Chinese hamster ovary cells are treated with melatonin; nor is neurite formation in neurobiastoma cells in culture affected by melatonin. It is concluded that melatonin does not interact with microtubules in a manner similar to colchicine and the Vinca alkaloids and it should not be classified as a colchicine-like mitotic inhibitor.

Quantitation of cytoplasmic fibrinogen in rat liver cells

Abstract Following an inflammatory response, the fibrinogen content of postmicrosomal supernatants from rat liver was quantitated using an enzyme-linked immunosorbent assay. A fivefold increase in intracellular fibrinogen was observed in the liver cells of turpentine-stimulated animals. The utility of the enzyme-linked immunosorbent assay for investigating the induction of fibrinogen synthesis was demonstrated.

Studies on the effect of the hepatocyte-stimulating factor on galactose-β1 → 4-N-acetylglucosamine α2 → 6-sialyltransferase in cultured hepatocytes

Abstract Rat hepatic Galβ1 → 4GlcNAcα2 → 6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Galβ1 → 4GlcNAcα2 → 6 isozyme.

Cystic fibrosis: The ciliary inhibitor is a small polypeptide associated with immunoglobulin G

Abstract A small polypeptide within the molecular weight range of 6,000 to 11,000 was found in immunoglobulin fractions from sera of cystic fibrosis homozygotes and heterozygotes. This factor appears to be responsible for interfering with ciliary activity in oyster gills. Since it can be dissociated from IgG without reductive cleavage it must be bound in a non-covalent manner. After its dissociation the IgG fractions from cystic fibrosis sera no longer inhibited ciliary activity. This finding explains the differences previously observed in the molecular weights of the ciliary inhibitor synthesized by cultured cells and that of sera from cystic fibrosis genotypes.

Comparison of albumin and fibrinogen biosynthesis in stimulated rats and cultured fetal rat hepatocytes

Abstract Evidence is presented that cultured fetal rat hepatocytes, when incubated with a crude leucocytic extract derived from rabbits undergoing an inflammatory response, show a marked increase in fibrinogen production and a concomitant decrease in albumin production. Antibody binding to polyribosomes synthesizing fibrinogen and albumin shows the same inverse relationship in rats undergoing an artificially induced inflammatory response. These data demonstrate directly and unequivocally that during the acute phase (inflammatory) response, the biosynthesis of these two plasma proteins is inversely affected by a factor or factors acting directly on the liver.

Changes in the extent of microtubule assembly can regulate initiation of DNA synthesis.

We have shown that MT depolymerization by colchicine and other drugs is sufficient to initiate DNA synthesis in serum-free cultures of embryonic fibroblasts and that stabilization of MTs with taxol inhibits this initiation. Growth factors and oncogenic DNA viruses also initiate DNA synthesis by a taxol-sensitive mechanism that appears to require MT depolymerization or rearrangements. Because we have shown that microtubule heterogeneity exists within single fibroblastic cells, we have carried out a series of experiments to determine the extent of microtubule disruption necessary to initiate DNA synthesis. We have compared the effects of various concentrations of colchicine and taxol on initiation of DNA synthesis with their effects on cytoplasmic MT complexes as visualized by indirect immunofluorescence microscopy and quantitated by direct binding of radiolabeled monoclonal antibody to cytoskeletons. The opposing effects of these drugs on MTs shows that there is a correlation between the extent of MT depolymerization and initiation of DNA synthesis. Initiation of DNA synthesis by colchicine in the presence of taxol is half-maximal when taxol and colchicine are added to cultures at a ratio of about 13 to 1. At this drug ratio, taxol stabilizes MTs near the nucleus, but MTs near the cell periphery are depolymerized. Maximal inhibition of DNA synthesis by taxol occurs only at taxol to colchicine ratios where MTs extend throughout the cytoplasm to the cell periphery. Thus, depolymerization of a small fraction of total MTs, particularly those near the periphery, may be sufficient to initiate proliferative events.