Ester M. Weijers

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.

TAFI and pancreatic carboxypeptidase B modulate in vitro capillary tube formation by human microvascular endothelial cells

Objective— Besides having a key role in fibrinolysis, the plasminogen system has been implicated in cell migration and angiogenesis. A common mechanism is the binding of plasminogen to carboxy-terminal lysine residues in partially degraded fibrin or on cellular surfaces. Here we examined the involvement of thrombin activatable fibrinolysis inhibitor (TAFI) and pancreatic carboxypeptidase B (CPB) in an in vitro capillary tube formation system, which is largely plasminogen-dependent. Methods and Results— Human microvascular endothelial cells (hMVECs) were seeded on a 3D plasma clot matrix and subsequently stimulated with bFGF/tumor necrosis factor (TNF)-&agr;. Tube formation was analyzed and fibrin degradation products (FbDP) were determined in the medium. Supplementation of the matrix with additional TAFI or CPB produced a reduction in tube formation. Pretreatment of hMVECs with CPB before seeding resulted in a similar effect. FbDP-levels indicated a concomitant reduction in matrix proteolysis. A TAFIa inhibitor increased tube formation and FbDP release into the medium. In separate assays, CPB impaired the migration of hMVECs in a dose-dependent manner, whereas proliferation and adhesion remained unaffected. Conclusions— Overall, these results demonstrate that TAFI and CPB in these systems modulate the plasminogen system both in the matrix and on the cell surface, thus leading to the inhibition of endothelial cell movement and tube formation.

Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities

Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches.

Immune-competent human skin disease models

All skin diseases have an underlying immune component. Owing to differences in animal and human immunology, the majority of drugs fail in the preclinical or clinical testing phases. Therefore animal alternative methods that incorporate human immunology into in vitro skin disease models are required to move the field forward. This review summarizes the progress, using examples from fibrosis, autoimmune diseases, psoriasis, cancer and contact allergy. The emphasis is on co-cultures and 3D organotypic models. Our conclusion is that current models are inadequate and future developments with immune-competent skin-on-chip models based on induced pluripotent stem cells could provide a next generation of skin models for drug discovery and testing.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

Introduction Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. Results The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. Conclusion The presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.

Methods to study differences in cell mobility during skin wound healing in vitro

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing.

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.

Extensive Characterization and Comparison of Endothelial Cells Derived from Dermis and Adipose Tissue: Potential Use in Tissue Engineering.

Tissue-engineered constructs need to become quickly vascularized in order to ensure graft take. One way of achieving this is to incorporate endothelial cells (EC) into the construct. The adipose tissue stromal vascular fraction (adipose-SVF) might provide an alternative source for endothelial cells as adipose tissue can easily be obtained by liposuction. Since adipose-EC are now gaining more interest in tissue engineering, we aimed to extensively characterize endothelial cells from adipose tissue (adipose-EC) and compare them with endothelial cells from dermis (dermal-EC). The amount of endothelial cells before purification varied between 4–16% of the total stromal population. After MACS selection for CD31 positive cells, a >99% pure population of endothelial cells was obtained within two weeks of culture. Adipose- and dermal-EC expressed the typical endothelial markers PECAM-1, ICAM-1, Endoglin, VE-cadherin and VEGFR2 to a similar extent, with 80–99% of the cell population staining positive. With the exception of CXCR4, which was expressed on 29% of endothelial cells, all other chemokine receptors (CXCR1, 2, 3, and CCR2) were expressed on less than 5% of the endothelial cell populations. Adipose-EC proliferated similar to dermal-EC, but responded less to the mitogens bFGF and VEGF. A similar migration rate was found for both adipose-EC and dermal-EC in response to bFGF. Sprouting of adipose-EC and dermal-EC was induced by bFGF and VEGF in a 3D fibrin matrix. After stimulation of adipose-EC and dermal-EC with TNF-α an increased secretion was seen for PDGF-BB, but not uPA, PAI-1 or Angiopoietin-2. Furthermore, secretion of cytokines and chemokines (IL-6, CCL2, CCL5, CCL20, CXCL1, CXCL8 and CXCL10) was also upregulated by both adipose- and dermal-EC. The similar characteristics of adipose-EC compared to their dermal-derived counterpart make them particularly interesting for skin tissue engineering. In conclusion, we show here that adipose tissue provides for an excellent source of endothelial cells for tissue engineering purposes, since they are readily available, and easily isolated and amplified.

Adipose tissue-derived stromal cells acquire endothelial-like features upon reprogramming with SOX18

Adipose tissue-derived stromal cells (ASC) form a rich source of autologous cells for use in regenerative medicine. In vitro induction of an endothelial phenotype may improve performance of ASCs in cardiovascular repair. Here, we report on an in vitro strategy using direct reprogramming of ASCs by means of ectopic expression of the endothelial-specific transcription factor SRY (sex determining region Y)-box18 (SOX18). SOX18 induces ASCs to express a set of genes involved in vascular patterning: MMP7, KDR, EFNB2, SEMA3G and CXCR4. Accordingly, SOX18 transduced ASCs reorganize under conditions of shear stress, display VEGF-induced chemotaxis and form tubular structures in 3D matrices in an MMP7-dependent manner. These in vitro findings provide insight into molecular and cellular processes downstream of SOX18 and show that reprogramming using SOX18 is sufficient to induce several endothelial-like features in ASCs.

Multiple Roles of Proteases in Angiogenesis

Proteases play an important role in various aspects of angiogenesis. First, pericellular proteolysis facilitates matrix degradation as well as migration and invasion of capillary sprouts into its surrounding matrix. (Membrane-type) matrix metalloproteases [(MT-)MMPs], ADAM(TS) metalloproteases, serine proteases, their inhibitors, and occasionally cysteine proteases are important players in pericellular proteolysis. The specific location and short exposure of protease activity can be explained by a tread-milling model with internalization of protease–receptor complexes as was proposed for MT1-MMP/MMP2 and urokinase/urokinase receptor complexes. Second, proteolytic events play a role in regulating VEGF-induced sprouting, such as proteasomal degradation of hypoxia inducible factors (HIFs), Notch-mediated cell signaling, and turnover of VEGF receptors. Third, proteases can activate and modify growth factors and receptors and thus generate split products with new biological functions and contribute to cell recruitment. Proteases likely also contribute to formation of anastomoses, which is required to restore circulation. Finally, new biological functions are also acquired by generation of split products from and modifications of matrix proteins. They include angiogenesis-inhibiting matrikines, and modified fibrin forms with altered angiogenic properties, which may bear perspective for tissue engineering applications.