P. Koolwijk

Fibrin structure and wound healing

Summary  Fibrinogen and fibrin play an important role in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, angiogenesis, and neoplasia. The contribution of fibrin(ogen) to these processes largely depends not only on the characteristics of the fibrin(ogen) itself, but also on interactions between specific‐binding sites on fibrin(ogen), pro‐enzymes, clotting factors, enzyme inhibitors, and cell receptors. In this review, the molecular and cellular biology of fibrin(ogen) is reviewed in the context of cutaneous wound repair. The outcome of wound healing depends largely on the fibrin structure, such as the thickness of the fibers, the number of branch points, the porosity, and the permeability. The binding of fibrin(ogen) to hemostasis proteins and platelets as well as to several different cells such as endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and keratinocytes is indispensable during the process of wound repair. High‐molecular‐weight and low‐molecular‐weight fibrinogen, two naturally occurring variants of fibrin, are important determinants of angiogenesis and differ in their cell growth stimulation, clotting rate, and fibrin polymerization characteristics. Fibrin sealants have been investigated as matrices to promote wound healing. These sealants may also be an ideal delivery vehicle to deliver extra cells for the treatment of chronic wounds.

Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells

Doxycycline is a commonly used broad-spectrum antibiotic. Recently, it has been shown that it also inhibits the activity of mammalian collagenases and gelatinases, an activity unrelated to its antimicrobial efficacy. In this study, we show that doxycycline not only inhibits MMP-8 and MMP-9 (gelatinase B) activity, but also the synthesis of MMPs in human endothelial cells. Doxycycline (50 μM) completely inhibited the phorbol-12-myristate-13-acetate (PMA)-mediated induction of MMP-8 and MMP-9, as measured by Western blotting and gelatin zymography, respectively. The inhibition was also observed at the mRNA level. No effect was observed on the expression of MMP-2 and of the MMP inhibitors TIMP-1 and TIMP-2. Chemically modified tetracyclines (CMTs) showed an inhibition similar to that of doxycycline, albeit less efficient. These observations demonstrate that endothelial cells display a specific regulation of MMPs, which may have implications for the pharmaceutical interaction in angiogenesis and angiogenesis-related diseases.

Angiopoietin-2, permeability oedema, occurrence and severity of ALI/ARDS in septic and non-septic critically ill patients

Background: Angiopoietin-2 and vascular endothelial growth factor (VEGF) may impair vascular barrier function while angiopoietin-1 may protect it. It was hypothesised that circulating angiopoietin-2 is associated with pulmonary permeability oedema and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) during septic or non-septic critical illness. Methods: Plasma levels of angiopoietin-1 and angiopoietin-2 were measured in mechanically ventilated patients (24 with sepsis, 88 without sepsis), together with the pulmonary leak index (PLI) for 67-gallium-labelled transferrin and extravascular lung water (EVLW) by transpulmonary thermal-dye dilution as measures of pulmonary permeability and oedema, respectively. ALI/ARDS was characterised by consensus criteria and the lung injury score (LIS). Plasma VEGF and von Willebrand factor (VWF) levels were assayed. Results: Angiopoietin-2, VWF, PLI, EVLW and LIS were higher in patients with sepsis than in those without sepsis and higher in patients with ALI/ARDS (n = 10/12 in sepsis, n = 19/8 in non-sepsis) than in those without. VEGF was also higher in patients with sepsis than in those without. Patients with high PLI, regardless of EVLW, had higher angiopoietin-2 levels than patients with normal PLI and EVLW. Angiopoietin-2 correlated with the PLI, LIS and VWF levels (minimum r = 0.34, p<0.001) but not with EVLW. Angiopoietin-2 and VWF were predictive for ARDS in receiver operating characteristic curves (minimum area under the curve = 0.69, p = 0.006). Angiopoietin-1 and VEGF did not relate to the permeability oedema of ALI/ARDS. Conclusion: Circulating angiopoietin-2 is associated with pulmonary permeability oedema, occurrence and severity of ALI/ARDS in patients with and without sepsis. The correlation of angiopoietin-2 with VWF suggests activated endothelium as a common source.

Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells.

Angiogenesis, the formation of new blood vessels from existing ones, plays a central role in development and in a number of pathological conditions. Tissue repair-associated angiogenesis usually involves cell invasion into a fibrin structure and the presence of inflammatory cells. In this chapter the role of plasminogen activators in the dissolution of fibrin and the invasion of endothelial cells into a fibrin matrix is described. Tissue-type plasminogen activator is stored in endothelial cells and can be released acutely into the vessel lumen upon stimulation of the endothelium to activate fibrinolysis and to prevent fibrin deposition. At the basolateral side of the cell, urokinase-type plasminogen activator (uPA) bound to a specific cellular receptor is involved in the proteolytic modulation of matrix proteins and cell-matrix interaction. The cytokine tumor necrosis factor-alpha (TNF-alpha) cooperates with the angiogenic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in inducing human microvascular endothelial cells in vitro to invade a three dimensional fibrin matrix and to form capillary-like tubular structures. The formation of these capillary-like tubules requires cell-bound uPA activity.

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

Summary.  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.

Blood outgrowth endothelial cells from cord blood and peripheral blood: angiogenesis-related characteristics in vitro

Summary.  Background: Blood outgrowth endothelial cells (BOEC) are good candidates for vascular (re‐) generating cell therapy. Although cord blood (CB) BOEC have been reported as more proliferative than peripheral blood (PB) BOEC, not much is known about their functional properties. Objectives: We have studied the following determinants in BOEC expanded from CB and PB: endothelial phenotype, in vitro adhesion, migration, proliferation, and angiogenic tube forming capacity. Methods/Results: Endothelial phenotype of BOEC was evaluated by fluorescence activated cell sorting (FACS) analysis and confirmed the presence of endothelial markers including CD31, CD105, CD144, CD146, KDR/VEGFR‐2, Tie‐2, and TNF‐α‐induced VCAM‐1 and ICAM‐1. Evaluation of cell proliferation revealed a higher basal proliferation of CB‐BOEC, which increased after exposure to bFGF but not VEGF. The lower basal proliferation of PB‐BOEC increased with VEGF or bFGF addition. Array analysis of angiogenic genes showed many comparable expressions in both BOEC, and a slightly more pronounced pro‐angiogenic profile in CB‐BOEC than PB‐BOEC. Both BOEC were able to form tubular structures in a three‐dimensional fibrin matrix. Tube formation in CB‐BOEC was markedly induced by TNF‐α only and inhibited by anti‐urokinase antibodies. It was comparable to that induced by combined addition of TNF‐α and VEGF or bFGF, while maximal tube formation in PB‐BOEC required simultaneous exposure to TNF‐α/VEGF or TNF‐α/bFGF. Conclusions: The endothelial phenotype and characteristics for homing, adhesion, migration, inflammation, and angiogenic tube formation are almost equal for BOEC from CB and PB. A slightly more angiogenic phenotype favors CB‐BOEC. However, addition of VEGF to PB‐BOEC induces equal proliferation and tube formation.

Molecular weight fibrinogen variants alter gene expression and functional characteristics of human endothelial cells.

Summary.  Background: Fibrin is a temporary matrix that not only seals a wound, but also provides a temporary matrix structure for invading cells during wound healing. Two naturally occurring fibrinogen variants, high molecular weight (HMW) and low molecular weight (LMW) fibrinogen, display different properties in supporting angiogenesis in vivo and in vitro. Objectives: This study was aimed at investigating the functional characteristics and molecular mechanisms of human microvascular endothelial cells (HMVECs) cultured on HMW and LMW fibrin matrices. Methods and results: HMVECs on HMW fibrin matrices showed increased proliferation and tube formation as compared with their counterparts on unfractionated and LMW fibrin. Degradation of HMW fibrin was markedly enhanced by the presence of HMVECs, that of LMW fibrin was enhanced only slightly. However, the expression levels of fibrinolysis‐regulating proteins and integrins were similar. Subsequent microarray analysis revealed that the expression of 377 genes differed significantly between HMVECs cultured on HMW fibrin and those cultured on LMW fibrin. Among these genes, UNC5B, DLL4 and the DLL4–Notch downstream targets Hey1, Hey2 and Hes1 showed increased expression in HMVECs on LMW fibrin. However, pharmacologic and genetic (DLL4 small interfering RNA) inhibition of DLL4–Notch signaling blunted rather than enhanced proliferation and tube formation by HMVECs on both fibrin variants. Conclusions: Heterogeneity in naturally occurring fibrinogen strongly influences endothelial cell proliferation and tube formation, and causes alterations in gene expression, including that of DLL4–Notch. The higher fibrinolytic sensitivity of HMW fibrin in the presence of HMVECs contributes to increased tube formation. Although the expression of DLL4–Notch was altered, it did not explain the enhanced tube formation in HMW fibrin. This study provides new perspectives for biological and tissue engineering applications.

Endothelial insulin receptor expression in human atherosclerotic plaques : linking micro- and macrovascular disease in diabetes?

OBJECTIVE Exogenous insulin use in patients with type 2 diabetes (DM2) has been associated with an increased risk of cardiovascular events. Through which mechanisms insulin may increase atherosclerotic plaque vulnerability is currently unclear. Because insulin has been suggested to promote angiogenesis in diabetic retinopathy and tumors, we hypothesized that insulin enhances intra-plaque angiogenesis. METHODS An in vitro model of pathological angiogenesis was used to assess the potential of insulin to enhance capillary-like tube formation of human microvascular endothelial cells (hMVEC) into a three dimensional fibrin matrix. In addition, insulin receptor expression within atherosclerotic plaques was visualized in carotid endarterectomy specimens of 20 patients with carotid artery stenosis, using immunohistochemical techniques. Furthermore, microvessel density within atherosclerotic plaques was compared between 68 DM2 patients who received insulin therapy and 97 DM2 patients who had been treated with oral glucose lowering agents only. RESULTS Insulin, at a concentration of 10(-8)M, increased capillary-like tube formation of hMVEC 1.7-fold (p<0.01). Within human atherosclerotic plaques, we observed a specific distribution pattern for the insulin receptor: insulin receptor expression was consistently higher on the endothelial lining of small nascent microvessels compared to more mature microvessels. There was a trend towards an increased microvessel density by 20% in atherosclerotic plaques derived from patients using insulin compared to plaques derived from patients using oral glucose lowering agents only (p=0.05). CONCLUSION Exogenous insulin use in DM2 patients may contribute to increased plaque vulnerability by stimulating local angiogenesis within atherosclerotic plaques.