Pieter Koolwijk

Role of fibrin matrix in angiogenesis.

Abstract: Angiogenesis, the formation of new blood vessels from existing vessels, plays an important role during development. In the adult, it is limited to the female reproductive system and to tissue repair and pathological conditions. Repair associated angiogenesis is usually accompanied by the presence of inflammatory cells, vascular leakage, and fibrin deposition. The temporary fibrin matrix acts, not only as a sealing matrix, but also as a scaffolding for invading leukocytes and endothelial cells during tissue repair. We have used a three‐dimensional fibrin matrix to study the outgrowth of human microvascular endothelial cells in capillary‐like tubular structures. This process is induced by the simultaneous addition of an angiogenic growth factor (bFGF or VEGF) and the cytokine TNFα, and is enhanced by hypoxia. It involves proteolytic activities, in particular cell bound urokinase/plasmin and matrix metalloproteinase activities. Modulation of the fibrin structure markedly affects the extent and stability of capillary tube formation in vitro. Preparation of fibrin at different pH (7.0–7.8) or crosslinking of the fibrin matrix induces differences in fibrin matrix rigidity and structure. This is accompanied by a change in capillary ingrowth. Heparins, in particular low molecular weight heparins, modulate the fibrin structure and by this action affect angiogenesis in vitro. A mutant fibrinogenNieuwegein, which lacks the terminal part of the Aα chain of fibrin harboring an RGD sequence and the transglutaminase sequence, provided additional evidence that the structure of fibrin is an important determinant for angiogenesis. These findings may have impact on improving wound healing and on influencing angiogenesis in malignancies with a fibrinous stroma.

Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions

BACKGROUNDnDecidual vascular development is important for implantation. This study analysed decidual vascular adaptation to implantation in correlation with miscarriage in decidual secretory endometrium (DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriage patients and matched controls.nnnMETHODSnDecidua was obtained during first trimester termination of pregnancy (controls) and vacuum aspiration in case of missed abortion (cases). Vascularization and the expression of VEGF-A, placental growth factor, Flt-1, KDR, angiopoietin (Ang)-1, Ang-2, TIE-2, and membrane-type matrix metalloproteinases MT1-, MT2-, MT3- and MT5-MMP were determined at mRNA and protein level. Uterine natural killer cells (CD56), macrophages (CD68), proliferation (Ki67) and apoptosis (activated caspase-3) were evaluated in consecutive sections.nnnRESULTSnDecidual vascularization showed differences between cases and controls, i.e. fewer vessels with larger circumference in cases. This correlated with the differential expressions of various factors at mRNA/antigen level and with increased endothelial flt1, KDR, MT2- and MT5-MMP expression in miscarriage patients. The differences between cases and controls were probably not based on altered proliferation and/or apoptosis, since Ki67 and active Caspase-3 showed comparable expression levels in both groups. Although DB of cases and controls showed similar amounts of CD56- and CD68-positive cells, the case group did show elevated levels of CD56 in DSE (P < 0.05) and of CD68 in DP compared with the control group (P < 0.05).nnnCONCLUSIONSnThe differences in vascularization and in the expression of angiogenic factors and proteases between groups suggest a correlation between decidual vascularization and the occurrence of miscarriages.

NF-κB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik−/− mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis.

Single and combined effects of αvβ3- and α5β1-integrins on capillary tube formation in a human fibrinous matrix

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-α and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the αvβ3-integrin inhibiting mAb LM609 or the α5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when αvβ3- and α5β1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. αvβ5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. αv-specific cRGD only inhibited angiogenesis when α5β1-integrin was simultaneously blocked. Endostatin mimicked the effect of α5β1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of αvβ3- and α5β1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.

Angiogenic Growth Factors and Their Receptors in First-Trimester Human Decidua of Pregnancies Further Complicated By Preeclampsia or Fetal Growth Restriction

Disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications. This study focused on the role of angiogenic factors in the first trimester in the pathogenesis of preeclampsia (PE) and/or fetal growth restriction (FGR). First-trimester decidua was obtained during chorionic villous sampling.The expression of the angiogenic factors was determined by reverse transcriptase polymerase chain reaction and related to the pregnancy outcome. First-trimester decidua expressed all angiogenic factors.The differential expression of angiogenic factors appeared to be more prominent in FGR than in PE. These first-trimester samples provided a unique opportunity to obtain information regarding the onset of PE and FGR. First-trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. This, in turn, may unintentionally set the stage for increased angiogenesis and altered decidual/placental vascular adaptation, which may be part of the pathogenesis of PE and/or FGR.

Endothelial plasminogen activators and matrix metalloproteinases in fibrinolysis and local proteolysis

Fibrin is a temporary matrix, which is formed after wounding of a blood vessel and when plasma leaks from blood vessels forming a fibrous exudate, often seen in areas of inflammation and in tumors.1 The fibrin matrix acts as a barrier preventing further blood loss, and provides a scaffolding in which new microvessels can infiltrate during wound healing. A proper timing of the outgrowth of microvessels, angiogenesis, as well as the subsequent (partial) disappearance of these vessels is essential to ensure adequate wound healing and to prevent the formation of scar tissue. It is generally believed that plasminogen activators play an important role in the migration of leukocytes and endothelial cells, and in the dissolution of the fibrin matrix.2,3 Plasminogen activators are serine proteases, which enzymatically convert the zymogen plasminogen into the active protease plasmin, the prime protease that degrades fibrin.

Role of Endothelial Plasminogen Activators in Fibrinolysis and Repair-Associated Angiogenesis

Fibrin is a temporary matrix, which is formed after wounding a blood vessel and when plasma leaks from blood vessels forming a fibrous exudate, often seen in areas of inflammation and in tumors (Dvorak et al., 1992). The fibrin matrix not only acts as a barrier preventing further blood loss, but also provides a structure in which new microvessels can infiltrate during wound healing. Proper timing of the outgrowth of microvessels as well as the subsequent (partial) disappearance of these vessels is essential to ensure adequate wound healing and to prevent the formation of scar tissue. It is generally believed that plasminogen activators play an important role in the migration and invasion of leukocytes and endothelial cells, and in the dissolution of the fibrin matrix (Kwaan, 1966; Pepper et al., 1990; Koolwijk et al., 1996). Plasminogen activators are serine proteases, which enzymatically convert the zymogen plasminogen into the active protease plasmin, the prime protease that degrades fibrin. The production of plasminogen activators by endothelial cells not only contributes to the proteolytic events related to the formation of microvessels in a wound, but also plays a crucial role in the prevention of thrombosis. If fibrin becomes deposited within the lumen of a blood vessel, cessation of the blood flow may occur accompanied by ischemia and eventually death of the distal tissues. The endothelium contributes considerably to the maintenance of blood fluidity by exposing anticoagulant molecules, by providing factors that interfere with platelet aggregation, and by its ability to stimulate fibrinolysis. Lysis of intravascularly generated fibrin must occur rapidly. However, it should be limited to a local area, because a general elevation of fibrinolysis upon wounding would result in recurrent bleeding.

Proteases and Angiogenesis. Regulation of Plasminogen Activators and Matrix Metalloproteases by Endothelial Cells

Angiogenesis, the outgrowth of new blood vessels from existing ones, is an essential process during development, but this normally stops when the body becomes adult. In the absence of injury, overt angiogenesis in adults is limited to the reproductive system of females (formation of corpus luteum and placenta). However, the formation of new blood vessels is an essential factor in tissue repair (formation and regression of granulation tissue), which is necessary to restore healthy tissue after wounding and/or inflammation, and is associated with many pathological conditions, such as tumor development and rheumatoid arthritis (Folkman and Klagsbrun, 1987; Liotta et al., 1991; Folkman and Shing, 1992; Montesano, 1992; Colville-Nash and Scott 1992). Fibrin (Dvorak et al., 1992), inflammatory cells (Polverini, 1989) and angiogenic factors (Broadley et al 1989; Klagsburn and D’Amore, 1991; Shweiki et al., 1992; Plate et al., 1992; Sengeret al., 1993; Koch et al., 1994) are commonly observed in angiogenesis associated with disease in man. A series of sequential events can be distinguished during the formation of new microvessels: (i) degradation of the vascular basement membrane and the fibrin or interstitial matrix by endothelial cells; (ii) endothelial cell migration; (iii) endothelial proliferation; and (iv) the formation of new capillary tubes and a new basement membrane (Folkman, 1986).

Plasminogen Activators in Fibrinolysis and Pericellular Proteolysis. Studies on Human Endothelial Cells in Vitro

Fibrin is a temporary matrix, which is formed after wounding of a blood vessel and when plasma leaks from blood vessels forming a fibrous exudate, often seen in areas of inflammation and in tumors.1 The fibrin matrix acts as a barrier preventing further blood loss, and provides a scaffolding in which new microvessels can infiltrate during wound healing. The outgrowth of new blood vessels from existing ones, angiogenesis, is an essential process during development, but normally stops when the body becomes adult. The half life time of endothelial cells in the adult body varies between 100 to 10,000 days in normal tissues, whereas it is reduced to several days in placenta an tumors.2 With the exception of the female reproductive system, angiogenesis in the adult is associated with tissue repair after injury by wounding or inflammation. Hence, in contrast to developmental angiogenesis, which proceeds independently of inflammation or fibrin deposition, adult angiogenesis is usually accompanied by the presence of fibrin and inflammatory cells or mediators. A proper timing of the outgrowth of microvessels as well as the subsequent (partial) disappearance of these vessels is essential to ensure adequate wound healing and to prevent the formation of scar tissue. Although essential for the formation of granulation tissue and tissue repair, angiogenesis, once under control of pathological stimuli, can contribute to a number of pathological conditions, such as tumor neovascularisation, pannus formation in rheumatoid arthritis, and diabetic retinopathy. Understanding the mechanisms involved in angiogenesis may provide clues to prevent pathological angiogenesis without seriously impairing tissue repair.

Production of Plasminogen Activators and Matrix Metalloproteinases by Endothelial Cells: Their Role in Fibrinolysis and Local Proteolysis

Matrices composed of fibrous proteins play an important role in the growth and repair of tissues. The complex extracellular matrix not only anchors the cell in the body, but it also provides the cell - via interaction with receptors and the cytoskeleton - with information about its position in the tissue. Various proteases, in particular plasmin, plasminogen activators (PAs) and matrix degrading metalloproteinases (MMPs) are involved in the regulation of the turnover and destruction of such matrices [Daneo et al., 1985; Saksela, 1985; Mignatti et al., 1986; Liotta et al., 1991].