Dermal collagen matrices for ventral hernia repair: comparative analysis in a rat model.

PURPOSE: The purpose of this study was to compare inflammatory responses, tissue integration, and strength of the acellular dermal collagen matrices AlloDerm(®)* Regenerative Tissue Matrix, Permacol™**Surgical Implant (Permacol), and CollaMend™*** Implant in a rat model for ventral hernia repair. METHODS: Rats were randomized into four groups and abdominal wall defects repaired with an inlay graft of AlloDerm, Permacol, or CollaMend. Rats were sacrificed at six time points and the defect area was removed and analyzed for tissue integration and physical strength. RESULTS: Variable cell infiltration was seen for the three implant groups. At of the all time points examined, cellular infiltration was most rapid in the AlloDerm implants and slowest for CollaMend. At 14 days, significant cell infiltration along with putative blood vessel formation was observed for AlloDerm, while Permacol implants exhibited a moderate level of infiltration. Very few cells penetrated CollaMend implants at 2 weeks. Cells had reached the center of the Permacol implants by 1 month, whereas CollaMend implants were encapsulated with a loose coat of disconnected cells, with very few cells infiltrating past the surface. At 6 months, AlloDerm and Permacol had evidence of cell penetration throughout the implants, while the CollaMend samples exhibited limited infiltration. Animals for each implant developed seromas: AlloDerm 40%, Permacol 33%, and CollaMend 83%. Mechanical testing revealed that AlloDerm at 6 months showed the lowest tensile strength, CollaMend the highest, and Permacol an intermediate level. CONCLUSIONS: The three biologics exhibited different patterns and rates of cellular and vascular permeation in our rat model. AlloDerm implants exhibited the most rapid and extensive cellular infiltration, followed by Permacol. However, on gross examination, the AlloDerm implants thinned significantly by 6 months. In contrast, the Permacol and CollaMend implants appeared to be largely intact.

TGF-beta signaling in A549 lung carcinoma cells: lipid second messengers.

Transforming growth factor-beta (TGF-beta) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF-beta in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF-beta inducible reporter construct, p3TP-Lux. Previous work from this laboratory using pharmacologic agents suggested that a phosphatidylcholine-specific phospholipase C and protein kinase C may be involved in early aspects of TGF-beta signaling. Here we provide evidence that TGF-beta induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP-Lux reporter plasmid are simultaneously treated with TGF-beta and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF-beta alone. We also find elevated levels of phosphocholine in cells following TGF-beta treatment. Further, exogenously added bacterial phosphatidylcholine phospholipase C (PC-PLC) is capable of inducing expression of the p3TP-Lux reporter to the same extent as TGF-beta indicating that the bacterial PC-PLC can mimic the TGF-beta effect. In contrast, neither hexanoyl sphingosine (a ceramide analogue) nor arachadonic acid induce expression of the p3TP-Lux reporter. Measurements with the fluorescent, calcium-sensitive dye, FURA2, indicated that there was no change in intracellular calcium in response to TGF-beta. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF-beta induction of the p3TP-Lux reporter. Thus the TGF-beta signaling pathway appears to involve the production of diacylglycerol but is independent of calcium.

Evidence for involvement of phosphatidylcholine-phospholipase C and protein kinase C in transforming growth factor-beta signaling.

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that elicits a wide variety of responses in cells. TGF-beta binds to cell surface receptors that contain cytoplasmic serine/threonine kinase domains. Here we provide evidence that both phospholipase C and protein kinase C (PKC) are involved in the TGF-beta activation of transcription and luciferase expression from the p3TP-Lux plasmid. Down-regulation of PKC prevents TGF-beta 1 induction of luciferase expression. Staurosporin and Calphostin C, inhibitors of PKC, block the ability of TGF-beta 1 to initiate transcription of the luciferase gene. Further, D609, an inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), and secondarily PKC also blocks TGF-beta 1-induced transcription of the transgene in A549 cells while the phosphatidylinositol-PLC pathway inhibitor U73122 is without effect. TGF-beta elevates steady-state mRNA levels for the endogenous PAI-1 and fibronectin genes. Treatment of cells with calphostin C or D609 prevents the TGF-beta-induced increase in these mRNAs. Together, these results suggest that PC-PLC and PKC are in a TGF-beta signaling pathway that results in elevated gene expression.

TGF beta alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype.

This study examines the mechanism by which TGF-beta 1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-beta 1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-beta 1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-beta 1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and alpha 1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-beta 1 response throughout the course of osteoblast differentiation. The initiation of a TGF-beta 1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-beta 1. These in vitro results indicate that TGF-beta 1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml.

Transforming growth factor-beta 1 induces expression of statin during differentiation of human promonocytic leukemia cells.

Transforming growth factor-Beta (TGF-beta) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP-1, we have shown that TGF-beta inhibits their proliferation and promotes differentiation into cells exhibiting macrophage-like properties. Therefore, a key question is whether TGF-beta influences the expression of genes associated with proliferation and/or growth inhibition. TGF-beta treatment of THP-1 cells results in downregulation of expression of c-myc. We also observe that TGF-beta 1-treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP-1 cells in response to TGF-beta 1 treatment. These results suggest that TGF-beta 1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells.

TGF-beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF-beta 1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF-beta 1 to affect the behavior of human THP-1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage-like cells. TGF-beta 1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF-beta 1-treated cells also express elevated levels of the beta 2 family of integrins. Taken together, these results suggest that TGF-beta 1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF-beta 1 and a myriad of other factors interact with many cell types to facilitate healing.

Responsiveness to transforming growth factor-beta (TGF-beta) restored by genetic complementation between cells defective in TGF-beta receptors I and II.

Selection of mutant Mv1Lu mink lung epithelial cells resistant to growth inhibition by transforming growth factor-beta (TGF-beta) has led to the isolation of cell clones with distinct alterations in type I and II TGF-beta receptors. Certain mutant clones present a decreased number or complete loss of detectable type I receptor. Other clones show a loss and/or altered electrophoretic mobility of the type II receptor, with concomitant loss of the type I receptor. Using somatic cell hybridization analysis we demonstrate the recessive nature of these mutants with respect to the wild-type phenotype and define various mutant complementation groups. Among these, hybrids between cells that express only type II receptor (R mutants) and cells that express neither receptor type (DRa mutants) rescue wild-type expression of type I receptors. Moreover, these hybrids regain full responsiveness to TGF-beta 1, as measured by inhibition of DNA synthesis as well as stimulation of fibronectin and plasminogen activator inhibitor-1 production. These results provide evidence for an interaction between TGF-beta receptor components I and II and show that, in Mv1Lu cells, expression of both receptor types is required for mediation of biological responses to TGF-beta 1.

Regulation of cell adhesion receptors by transforming growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit.

Cell adhesion to extracellular matrices is mediated by a set of heterodimeric cell surface receptors called integrins that might be the subject of regulation by growth and differentiation factors. We have examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of the very late antigens or alpha beta 1 group of integrins in human cell lines. The six known members of this family share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward type I collagen, fibronectin, laminin, and other as yet unknown cell adhesion proteins. Using a panel of specific antibodies and cDNA probes, we show that in WI-38 lung fibroblasts TGF-beta 1 elevates concomitantly the expression of alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits at the protein and/or mRNA level, their assembly into the corresponding alpha beta 1 complexes, and their exposure on the cell surface. The rate of synthesis of total alpha subunits relative to beta 1 subunit is higher in TGF-beta 1-treated cells than in control cells. The characteristically slow (t1/2 approximately 10 h) rate of beta 1 conversion from precursor form to mature glycoprotein in untreated cells increases markedly (to t1/2 approximately 3 h) in response to TGF-beta 1. The results suggest that in WI-38 fibroblasts the beta 1 subunit is synthesized in excess over alpha subunits, and assembly of beta 1 subunits with rate-limiting alpha subunits is required for transit through the Golgi and exposure of alpha beta 1 complex on the cell surface. TGF-beta 1 does not induce the synthesis of integrin subunits that are not expressed in unstimulated cells, such as alpha 4 and alpha 6 subunits in WI-38 fibroblasts. However, alpha 4 and alpha 6 subunits can be regulated by TGF-beta in those cells that express them. The results suggest that TGF-beta regulates the expression of individual integrin subunits by parallel but independent mechanisms. By modifying the balance of individual alpha beta 1 integrins, TGF-beta 1 might modulate those aspects of cell migration, positioning, and development that are guided by adhesion to extracellular matrices.

Regulation of cell adhesion receptors by transforming growth factor-beta. Regulation of vitronectin receptor and LFA-1.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) to regulate the expression of members of the alpha beta 2 and alpha beta 3 families of integrins. TGF-beta 1 elevates the expression of vitronectin receptors (alpha v beta 3 integrin) in all cells examined including WI-38 human lung fibroblasts, 3T3-L1 mouse fibroblasts, and MG-63 human osteogenic sarcoma cells. TGF-beta 1 action increases the level of mRNA and the synthesis of vitronectin receptor subunits with t1/2 o 3-4 h and 6 h, respectively. TGF-beta 1 up-regulates expression of the intercellular adhesion receptor, LFA-1 (alpha L beta 2), in THP-1 human monocytic leukemia cells by increasing the synthesis of alpha L subunit but not beta 2 subunit. The increase in alpha L synthesis and assembly into LFA-1 complexes induced by TGF-beta 1 occurs in parallel with elevated fibronectin receptor synthesis in THP-1 cells. These responses to TGF-beta 1 are lost upon phorbol ester-induced differentiation of THP-1 cells into the macrophage phenotype. The results suggest a role of TGF-beta in the regulation of cell-matrix interactions mediated by vitronectin receptors and cell-cell interactions mediated by LFA-1 in the immune system.

Cell adhesion protein receptors as targets for transforming growth factor-beta action.

Transforming growth factor-beta (TGF-beta) increases the incorporation of fibronectin and type I collagen into the extracellular matrix of fibroblasts and epithelial cells and enhances the attachment of thymocytes onto a fibronectin substratum. Investigation of the molecular basis for these effects showed that TGF-beta elevates specifically the expression of cell adhesion protein receptors. Treatment of cells with either form of TGF-beta, TGF-beta 1, or TGF-beta 2, increases the rate of receptor synthesis and the level of receptors on the cell surface. TGF-beta acts via two complementary mechanisms, elevation of receptor mRNA and faster kinetics of receptor beta subunit precursor to product conversion. The results show that the expression of cell adhesion receptors is susceptible to pretranslational and posttranslational regulation by factors that control cell morphology, proliferation, and differentiation such as TGF-beta.

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta.

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.

Multiple type-beta transforming growth factors and their receptors.

Type beta transforming growth factors are a group of homologous structurally related polypeptides that act on a wide variety of cell types to alter their proliferative and phenotypic properties. TGF-beta s form a group within a larger family of polypeptides that control developmental processes in organisms from humans to Drosophila. We have found that at least three distinct forms of TGF-beta are present in mammalian tissues. We have identified a family of cell surface glycoproteins that bind TGF-beta s with high affinity and specificity. Examination of the interactions between individual forms of TGF-beta and the individual TGF-beta receptor species has illustrated a complex pattern of ligand-receptor associations. Occupancy of a particular receptor type by TGF-beta can be correlated to the dictation of specific effects on cell proliferation and cell differentiation.

Biologically active precursor for transforming growth factor type alpha, released by retrovirally transformed cells.

Retrovirus-transformed rat cells that actively express transforming growth factor type alpha (TGF-alpha) release into the medium a soluble protein of 17-19 kDa that has been identified as a precursor for TGF-alpha. The identification of this protein as a TGF-alpha precursor is based on its recognition by specific antibodies and by the ability of elastase to convert this protein into a 6-kDa polypeptide with the properties of mature TGF-alpha. This TGF-alpha precursor binds to epidermal growth factor/TGF-alpha receptors and activates the receptor-associated tyrosine kinase activity in intact cells. The biological potency of this precursor is not markedly increased by conversion into mature TGF-alpha in vitro. These studies demonstrate the ability of transformed cells to release a TGF-alpha precursor capable of strong mitogenic action in vivo.

Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix.

We have examined whether the extracellular matrix is a biochemical target for transforming growth factor-beta (TGFbeta). We find that TGFbeta increases the expression of the major extracellular matrix proteins, fibronectin and collagen. This effect is a general response to TGFbeta seen in primary cultures and established lines of cells from various types, normal and transformed. The relative incorporation of fibronectin and collagen into the matrix also increases in response to TGFbeta. The effect of TGFbeta on fibronectin levels as characterized in chick embryo fibroblasts is rapid, selective, persistent, and specific, and involves transcriptional events; it is not mimicked by other growth factors tested. The induction of anchorage-independent growth of normal fibroblasts by TGFbeta is mimicked by fibronectin and is specifically blocked by inhibitors of fibronectin binding to its cell surface receptor. The results demonstrate a functional involvement of fibronectin in mediating cellular responses to TGFbeta, and suggest a model for TGFbeta action based on the control of the extracellular matrix in target cells.

Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.

Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts.

On the mechanism by which insulin stimulates protein synthesis in chick embryo fibroblasts.

It has been known that insulin raises the rate of incorporation of [3H]leucine into the total protein of hormone-deficient chick embryo fibroblasts by approximately 1.5-fold. The elevation is not dependent upon the production of new messenger ribonucleic acids (mRNAs). Evidence is now presented in support of the following points: the greater labelling is due to more rapid polypeptide synthesis, not to an increase in the specific activity of leucyl-tRNA; the enhanced synthesis derives largely or entirely from a speeding up of the process of initiation, rather than that of elongation or termination; and the 1.5-fold stimulation is due to the elevated rates of formation of at least many of the fibroblast proteins. The hormone was shown before to stimulate posttranscriptionally and highly preferentially for formation of ribosomal proteins in the resting chick embryo cells. The question has been asked here whether insulin increases the production of total cell ribosomal protein by chemically altering preformed mRNAs. Results obtained by translating messages from deprived and hormone-treated cells in wheat germ and reticulocyte preparations do not support a mechanism involving covalent modification of preformed mRNAs. The observations, coupled with those previously made with inhibitors of translation, lead us to suggest that insulin stimulates protein synthesis in the resting chick embryo cells by activating limiting components of the initiation system. The effects of the hormone are greatest with messages, such as those for the ribosomal proteins, that have low affinities for the limiting initiation components.

Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period.

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non-ribosomal RNAs: (1) Addition to the "shift-up" medium of either of two inhibitors of RNA formation, camptothecin or 5, 6-dichloro--1-beta-D-ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells through the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced. The possibility is considered that the essential G1 molecules are mRNAs.