Livingston Van De Water

Vascular permeability factor (VPF, VEGF) in tumor biology

SummaryVascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine expressed and secreted at high levels by many tumor cells of animal and human origin. As secreted by tumor cells, VPF/VEGF is a 34–42 kDa heparin-binding, dimeric, disulfide-bonded glycoprotein that acts directly on endothelial cells (EC) by way of specific receptors to activate phospholipase C and induce [Ca2+]i transients. Two high affinity VPF/VEGF receptors, both tyrosine kinases, have thus far been described. VPF/VEGF is likely to have a number of important roles in tumor biology related, but not limited to, the process of tumor angiogenesis. As a potent permeability factor, VPF/VEGF promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. This matrix promotes the ingrowth of macrophages, fibroblasts, and endothelial cells. Moreover, VPF/VEGF is a selective endothelial cell (EC) growth factorin vitro, and it presumably stimulates EC proliferationin vivo. Furthermore, VPF/VEGF has been found in animal and human tumor effusions by immunoassay and by functional assays and very likely accounts for the induction of malignant ascites. In addition to its role in tumors, VPF/VEGF has recently been found to have a role in wound healing and its expression by activated macrophages suggests that it probably also participates in certain types of chronic inflammation. VPF/VEGF is expressed in normal development and in certain normal adult organs, notably kidney, heart, adrenal gland and lung. Its functions in normal adult tissues are under investigation.

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device.

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero (“cliff” gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant (“hill” gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.

Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition

Tumor stroma formation results from the interaction of tumor cells and their products with the host and certain of its normal defense mechanisms, particularly the clotting and fibrinolytic systems. It is a process in which tumor cells render local venules and veins hyperpermeable with the result that fibrinogen and other proteins extravasate and clot, forming an extravascular crosslinked fibrin gel. Coagulation is mediated by an interaction between extravasated plasma clotting factors and tumor-associated and perhaps other tissue procoagulants. Parallel activation of the fibrinolytic system leads to substantial fibrin turnover, but fibrin nonetheless accumulates in amounts, variable from tumor to tumor, that are sufficient to provide a provisional stroma. This provisional stroma imposes on tumor cells a structure that persists even as tumor cells multiply and as the fibrin provisional stroma is replaced by mature connective tissue. The provisional fibrin stroma also serves to regulate the influx of macrophages, and perhaps other inflammatory cells, but at the same time, and in ways that are not fully understood, facilitates the inward migration of new blood vessels and fibroblasts, integral components of mature tumor stroma. Ascites tumors differ from solid tumors in that fibrin gel is not ordinarily deposited in body cavities and, as a result, there is no provisional stroma to impose an initial structure. Tumor stroma generation resembles the process of wound healing in many respects. However, it differs in the mechanism of its initiation, and in the apparent lack of a role for platelets. It also differs fundamentally in that invading tumor cells continually render new vessels hyperpermeable to plasma, thus perpetuating the cycle of extravascular fibrin deposition. In this sense, tumors behave as wounds that do not heal. Largely neglected in this review has been discussion of the numerous cytokines, mitogens, and growth factors that are widely believed to play important roles in tumor angiogenesis and wound healing; i.e., PDGF, FGF, EGF, TGF alpha, TGF beta, TNF, interferons, etc. This omission has been intentional, and for two reasons. First, these cytokines have already received considerable attention [100,123-128]. Second, it is not yet clear how closely the actions of these molecules, as described in vitro, relate to their functions in vivo. At present we are deluged with a surfeit of factors that have the capacity to induce new blood vessel formation in angiogenesis assays; these factors include not only peptides but lipids and even ions [126,129-131].(ABSTRACT TRUNCATED AT 400 WORDS)

Vascular Permeability Factor/Vascular Endothelial Growth Factor: An Important Mediator of Angiogenesis in Malignancy and Inflammation

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine that is overexpressed in many transplantable animal and autochtonous human cancers, in healing wounds, and in chronic inflammatory disorders such as psoriasis and rheumatoid arthritis. All of these entities are characterized by angiogenesis, altered extracellular matrix, and variable degrees of hypoxia. In addition, two VPF/VEGF receptors, flt-1 and kdr, are overexpressed by endothelial cells that line the microvessels that supply these tumors/inflammatory reactions. On the basis of these and other data, we have proposed a model of angiogenesis in which VPF/VEGF plays a central role: this model is applicable to tumors and also to the angiogenesis that occurs in non-neoplastic processes.

Influence of insulin therapy on burn wound healing in rats

BACKGROUND Insulin is proposed as a therapy for suppressing muscle wasting after burn trauma although the long-term effects of this therapy on wound healing are not yet known. The present study was designed to investigate the effect of systemically administered insulin therapy on burn wound healing. MATERIALS AND METHODS Young rats weighing 80-150 g were subjected to 15-20% total body surface area burn injury on their shaved dorsum. The insulin dosage was increased over the first 3 days in each rat from 0.25 U (Day 1), 0.5 U (Day 2), and 1.0 U (Day 3) per 100 g body wt. The rats were euthanized at the fourth or fifteenth day postinjury. Skin sections were analyzed by histochemistry and quantitative polarization microscopy. RESULTS Histology showed a decreased number of inflammatory cells and increased vasodilation in the insulin-treated animals at Day 4 relative to untreated rats; at Day 15 there was increased reepithelialization. Quantitative analysis using polarization microscopy and picrosirius red staining showed an increased collagen deposition in wounds by Day 4 in insulin-treated rats relative to untreated burn controls. CONCLUSION These results indicate that insulin induces accelerated wound healing associated with diminished inflammation and increased collagen deposition.

Mechanisms by which fibrin and fibronectin appear in healing wounds : Implications for Peyronie's disease

ABSTRACTPurpose: The extracellular matrix that is present at sites of tissue repair in most instances undergoes an orderly transition from a fibrin containing matrix to collagen-rich scar. However, in some conditions, such as Peyronies disease, fibrin persists. Evidence from a number of experimental systems indicates that extracellular matrix proteins and their receptors serve an important function in regulating cell behaviors. Thus, the presence of the fibrin matrix is likely to have important implications in the course of either normal or pathogenic wound healing as occurs in Peyronies disease. Potential mechanisms by which the fibrin rich provisional matrix appears in healing wounds are presented.Materials and Methods: Methodologies, such as in situ hybridization, immunolocalization and labeled tracer techniques, were used in our study.Results: We found by these approaches that the 2 general mechanisms that contribute to the generation of the wound extracellular matrix are leakage of plasma proteins,...

Balloon Catheterization Induces Arterial Expression of Embryonic Fibronectins

Fibronectins (FNs) comprise a family of adhesive extracellular matrix proteins that arise by alternative splicing in three regions: V (IIICS), EIIIA (ED-A), and EIIIB (ED-B). FNs bearing the EIIIA and EIIIB segments are prevalent during embryogenesis, expressed to lesser degrees in normal adult tissues, and may be locally reexpressed at adult tissue injury. RNase mapping shows that normal rat arteries express low levels of FNs that are predominantly EIIIA- and EIIIB-. Following balloon injury, arterial walls produce increased total levels of FN transcripts that preferentially include both the EIIIA and EIIIB segments. However, despite inducing increased total FN mRNA, balloon injury does not alter the relative composition of V120+, V95+, AND V0 spliced forms. In situ hybridization reveals that as early as 4 days after injury medial cells express increased total FN mRNA, and by 7 days substantial neointimal and focal medial synthesis of EIIIA+, EIIIB+, and V120+ FNs occurs; macrophages do not significantly contribute to this observed vascular FN synthesis. Consistent with the mRNA data, immunofluorescence microscopic analysis reveals increased deposition of EIIIB+ and V+ FN protein forms in injured arterial walls, particularly within the neointima. Our results suggest that local synthesis of specific FN isoforms is important to the neointimal formation that ensues after balloon injury.

Identification of Two Amino Acids within the EIIIA (ED-A) Segment of Fibronectin Constituting the Epitope for Two Function-blocking Monoclonal Antibodies

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell α-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-β-mediated smooth muscle cell α-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a β strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.

VEGF expression by epithelial and stromal cell compartments: resolving a controversy.

Vascular endothelial growth factor (VEGF) expression in healing wounds provokes dermal angiogenesis through complex mechanisms involving promotion of endothelial cell proliferation 1,2 and survival, 3-5 specific induction of endothelial cell gene expression, 6-10 and increased microvascular permeability. 11,12 Microvascular hyperpermeability transforms the extracellular matrix by promoting extravasation of fibrinogen, 13 fibronectin, 14 and other proteins from blood plasma. 14