Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo.

BACKGROUND: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. OBJECTIVE: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. RESULTS: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. CONCLUSIONS: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing.

Influence of plasma triglyceride and plasma cholesterol levels on the clearance rate of fibrinogen.

Increased plasma levels of fibrinogen are associated with a higher risk of cardiovascular disease. It has been suggested that lipid levels may influence the fibrinogen levels by a mechanism other than the synthesis rate, for example a decreased clearance rate. We performed a pilot study to explore this possibility. Twelve male Wistar rats were fed for four weeks with a control low fat/low cholesterol diet, a high fat/high cholesterol diet, and a high fat/high cholesterol diet with an additional 0.5% cholic acid. Labeled 125I fibrinogen was injected, and blood was sampled repeatedly. From the plasma radioactivity of the samples, fibrinogen halflife time was calculated for each animal. Our results suggest that plasma lipids lengthen the fibrinogen halflife times, although the differences were not statistically significant in this small study. Our final conclusion from this study is that lipids may have an effect on the turnover rate of fibrinogen and possibly affect fibrinogen levels through this mechanism.

Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells.

The underlying mechanism of the antiproliferative effect of S (simvastatin), a HMG-CoA reductase inhibitor, in vascular smooth muscle cells (SMC) is still poorly understood. In the present study, we used synchronized human SMC, isolated from left interior mammary artery, as an in vitro model to test the effects of S on platelet-derived growth factor (PDGF)-induced DNA synthesis, extracellular-regulated kinase 1/2 (ERK1/2), p38/stress-activated protein kinase 2 (SAPK2), RhoA and Rac1 activation. ERK1/2 phosphorylation was triggered within 2 min of PDGF stimulation (early G1 phase) and was blocked by PD98059, a specific inhibitor of the ERK1/2 pathway, which also strongly inhibited PDGF-induced DNA synthesis (IC(50) = 10 micromol/L). PDGF quickly induced p38 phosphorylation (early G1 phase) and SB203580, a specific inhibitor of the p38/SAPK2 pathway, also blocked PDGF-induced DNA synthesis (IC(50) = 0.3 micromol/L). Translocation to the plasma membrane of small GTPases, such as RhoA and Rac1, could not be detected within 15 min of stimulation with PDGF or lysophosphatidic acid (LPA) (early G1 phase), but occurred after 24 hr of PDGF stimulation (late G1/S phase). S inhibited PDGF-induced DNA synthesis (IC(50) = 3.5 micromol/L), and this effect was dependent on intracellular mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate availability. The critical time period for the reversal of the S effect by mevalonate comprised both the early and late G1 phase of the SMC cycle. PDGF-induced ERK1/2 phosphorylation and PDGF-induced p38 phosphorylation were not markedly affected by S during the whole G1 phase. However, S treatment blocked the PDGF- and LPA-induced membrane translocation of RhoA that occurred during the late G1/S phase. In the case of Rac1, the same process was also inhibited by S treatment. We concluded from these results that, in SMC, the early events associated with ERK1/2 and p38 signal transduction pathways, recruited for PDGF-mediated DNA synthesis, were insensitive to S action, whereas the mevalonate-dependent, posttranslational modification of RhoA and Rac1 molecules, required for PDGF-induced membrane translocation, was blocked by this drug. These results suggest that the antiproliferative effect of S can be explained not only by the blockage of RhoA-mediated signaling events but also by Rac1-mediated signaling events.

Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types.

The effects of 6 HMG-CoA reductase inhibitors: pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin were analyzed in cultured human smooth muscle cells, fibroblasts, endothelial cells and myoblasts. In vascular smooth muscle cells, pravastatin was a much weaker inhibitor of cholesterol synthesis than the 5 other drugs which displayed equally strong inhibitory potency. The anti-proliferative effects of these 6 drugs were analyzed by measuring cell number and mitochondrial dehydrogenase activity (MTT assay) after 3 days of incubation. IC25 values for inhibition of proliferation were very similar among the 4 cell types and were in the following order of magnitude: pravastatin << lovastatin = simvastatin = atorvastatin = fluvastatin << cerivastatin. Only in the case of pravastatin was proliferation inhibited at lower concentration in smooth muscle cells than in the other cell types. Proliferation was also assessed by measuring DNA synthesis in these cells. A 3 day-incubation with 1 microM of pravastatin had no effect on this parameter in all 4 cell types. However, 1 microM of simvastatin or lovastatin caused either an inhibition (in smooth muscle cells and endothelial cells) or stimulation (in fibroblasts) of this process. The effects of simvastatin on cell number, mitochondrial dehydrogenase activity and DNA synthesis were counteracted by simultaneous mevalonate addition. Simvastatin treatment was also associated with a change in the post-translational modification of the ras protein in smooth muscle cells, probably by inhibition of its farnesylation. Moreover, simvastatin treatment blocked the PDGF and bFGF-induced DNA synthesis in synchronized smooth muscle cells, whereas it does not affect the fetal calf serum-induced DNA synthesis in synchronized fibroblasts, suggesting that simvastatin blocks various steps of the cell cycle and that this effect depends on the cell type and the growth signalling pathway activated.

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Activated gelatinase-B (MMP-9) and urokinase-type plasminogen activator in synovial fluids of patients with arthritis. Correlation with clinical and experimental variables of inflammation.

OBJECTIVE: To evaluate the occurrence of gelatinase-B (matrix metalloproteinase 9, MMP-9) in synovial fluids (SF) of patients with arthritis to investigate the possible role of this neutral MMP in joint destruction. METHODS: In paired (series I) and unpaired SF (series II) we examined the occurrence of gelatinase-B, prostromelysin-1, and urokinase-type plasminogen activator (u-PA). RESULTS: In the paired SF a parallelism between the presence of activated gelatinase-B and the local arthritis activity scores of the knees was observed. Activated gelatinase-B correlated well with the presence of stromelysin-1 and u-PA, 2 enzymes probably involved in the activation process of gelatinase-B. In the 2nd series, activated gelatinase-B was found in 56 SF samples, whereas 82 samples did not exhibit activated gelatinase-B. The SF samples with the activated form of gelatinase-B showed a significantly higher ability to induce permeability changes in cultured monolayers of human endothelial cells, had more myeloperoxidase activity--secreted by infiltrated leukocytes--and had higher u-PA antigen concentrations, compared to SF samples without the activated form of gelatinase-B. CONCLUSION: Our data suggest that the presence of gelatinase-B is a reflection of the inflammatory condition of the joints of patients with arthritis, and that the activation of gelatinase-B in the joints, which may occur in a u-PA/plasmin dependent and/or a stromelysin dependent way, contributes to the progression of arthritis.

Resistance to collagen-induced arthritis in a nonhuman primate species maps to the major histocompatibility complex class I region.

Type II collagen-induced arthritis (CIA) is an experimentally inducible autoimmune disorder that is, just like several forms of human arthritis, influenced by a genetic background. Immunization of young rhesus monkeys (Macaca mulatta) with type II collagen (CII) induced CIA in about 70% of the animals. One major histocompatibility complex (MHC) class I allele was present only in young animals resistant to CIA and absent in arthritic animals. This strong association suggests that the MHC class I allele itself, or a closely linked gene, determines resistance to CIA. The mechanism controlling the resistance to CIA becomes less efficient in aged animals since older rhesus monkeys, which were positive for the resistance marker, developed a mild form of arthritis. At the cellular level it is demonstrated that resistance to CIA is reflected by a low responsiveness of T cells to CII. This association between a specified MHC class I allele and resistance to an autoimmune disease points at the importance of the MHC class I region in the regulation of the immune response to an autoantigen.

Acquired resistance to type II collagen-induced arthritis in rhesus monkeys is reflected by a T cell low-responsiveness to the antigen.

Ten out of 14 rhesus monkeys developed arthritis after a single immunization with bovine type II collagen (B-CII). In contrast to primary resistant monkeys, arthritic animals showed a B-CII specific T cell proliferation during the induction phase of the disease. All surviving animals showed a full remission of the disease. Two monkeys acquired resistance to collagen-induced arthritis (CIA) after one period of disease, but in three animals a booster immunization with B-CII induced a slight flare-up. It is demonstrated that B-CII immunized rhesus monkeys have the capacity to restore resistance to CIA. The development of resistance to CIA is reflected by a decreased T cell responsiveness to B-CII. It is shown that the lack of IL-2 plays a role in B-CII-induced T cell low-responsiveness. A potential role of CD8+ T cells in the down regulation of the T cell response to B-CII is discussed.

Collagen-induced arthritis in an outbred group of rhesus monkeys comprising responder and nonresponder animals. Relationship between the course of arthritis and collagen-specific immunity.

It is speculated that the autoimmune response to type II collagen (CII) is a driving force in the pathogenesis of human rheumatoid arthritis (RA). In this report, we describe the relationship between the induction of collagen arthritis and the CII-specific humoral, as well as cellular, immune response in rhesus monkeys. Ten of 14 monkeys immunized with bovine type II collagen (B-CII) developed polyarthritis. Susceptible animals showed a T cell response to B-CII; resistant animals did not. After the primary immunization, the humoral response to B-CII, as well as to rhesus monkey type II collagen, was dominated by antibodies of the IgM isotype in the susceptible animals and by antibodies of the IgG isotype in the resistant animals. Because of the close phylogenic relationship between the rhesus monkey and humans, these data contribute valuable information about the role of CII-specific immunity in the pathogenesis of human RA.

Experimental immune mediated arthritis in rhesus monkeys. A model for human rheumatoid arthritis?

The induction of experimental arthritis in rhesus monkeys was studied by intradermal immunization of bovine type II collagen and antigens derived from Mycobacterium tuberculosis, Streptococcus pyogenes, and Eubacterium aerofaciens. The tested bacterial antigens proved to be not arthrogenic. Bovine type II collagen induced clinical arthritis in 50% of the rhesus monkeys. Type II collagen induced arthritis in rhesus monkeys proved to be a potential model to study clinical, serological, histological, genetic, and immunologic features associated with human RA.