Joost O. Fledderus

KLF2 Primes the Antioxidant Transcription Factor Nrf2 for Activation in Endothelial Cells

Objective—Atheroprotective blood flow induces expression of anti-inflammatory Krüppel-like factor 2 (KLF2) and activates antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in vascular endothelium. Previously, we obtained KLF2-induced gene expression profiles in ECs, containing several Nrf2 target genes. Our aim was to investigate the role of KLF2 in shear stress–mediated activation of Nrf2 in human umbilical vein endothelial cells (HUVECs). Methods and Results—Expression of Nrf2 and its targets NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase (HO-1) was elevated by shear and KLF2. KLF2 knockdown showed that shear-induced expression of NQO1 but not Nrf2 was dependent on KLF2. KLF2 overexpression in absence of flow resulted in more efficient activation of Nrf2 by tert-butyl hydroquinone (tBHQ) through enhanced nuclear localization, and promoted expression of a large panel of Nrf2-dependent genes resulting in superior protection against oxidative stress. Comparison of shear-, KLF2-, and Nrf2-induced transcriptomes showed that the majority of shear-modulated gene sets is influenced by KLF2 or Nrf2. Conclusions—We report that KLF2 substantially enhances antioxidant activity of Nrf2 by increasing its nuclear localization and activation. The synergistic activity of these two transcription factors forms a major contribution to the shear stress–elicited transcriptome in endothelial cells.

Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin microparticles (GMPs) as a means to prolong VEGF activity at the preferred location within 3D bioprinted scaffolds, and the effects on subsequent vascularization. The release of VEGF from GMPs was continuous for 3 weeks during in vitro studies, and bioactivity was confirmed using human endothelial progenitor cells (EPCs) in migration assays. Traditional and real-time migration assays showed immediate and efficient EPC migration in the presence of GMP-released VEGF, indistinguishable from VEGF-solution that was added to the medium. Matrigel scaffolds containing EPCs and VEGF, which was released either in a fast or sustained fashion by application of GMPs, were investigated for their in vivo vasculogenic capacity. Implantation in subcutaneous pockets in nude mice for one week demonstrated that vessel formation was significantly higher in the VEGF sustained-release group compared to the fast release group. In addition, regional differences with respect to VEGF release were introduced in 3D bioprinted EPC-laden scaffolds and their influence on vasculogenesis was investigated in vivo. The different regions were retained and vessel formation occurred analogous with the results seen in the Matrigel plugs. We conclude that GMPs are suitable to generate sustained release profiles of bioactive VEGF, and that they can be used to create defined differentiation regions in 3D bioprinted heterogeneous constructs, allowing a new generation of smart scaffold design. The prolonged presence of VEGF led to a significant increase in scaffold vascularization when applied in vivo.

KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium

The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

KLF2 Suppresses TGF-β Signaling in Endothelium Through Induction of Smad7 and Inhibition of AP-1

Objective—The flow-responsive Kruppel-like factor 2 (KLF2) is crucial for maintaining endothelial cell quiescence. Here, we describe its detailed effects on transforming growth factor-&bgr; (TGF-&bgr;) signaling, which normally has proatherogenic effects on endothelium. Methods and Results—In-depth analysis of genome-wide expression data shows that prolonged lentiviral-mediated overexpression of KLF2 in human umbilical vein endothelial cells (HUVECs) diminishes the expression of a large panel of established TGF-&bgr;–inducible genes. Both baseline and TGF-&bgr;–induced expression levels of plasminogen activator inhibitor 1 (PAI-1) and thrombospondin-1 are greatly diminished by KLF2. Using a combination of ectopic expression, small interfering RNA–mediated knockdown, and promoter activity assays, we show that KLF2 partly inhibits the phosphorylation and subsequent nuclear accumulation of Smad2, thereby suppressing the TGF-&bgr;–induced Smad4-mediated transcriptional activity. This is achieved through TGF-&bgr;–independent induction of inhibitory Smad7. Additionally, a full inhibition of TGF-&bgr; signaling is functionally achieved through a simultaneous suppression of activator protein 1 (AP-1), which is an essential cofactor for TGF-&bgr;–dependent transcription of many genes. Conclusions—The concerted mechanism by which KLF2 inhibits TGF-&bgr; signaling through induction of inhibitory Smad7 and attenuation of AP-1 activity provides a novel mechanism by which KLF2 contributes to sustaining a quiescent, atheroprotective status of vascular endothelium.

Interferon-β Signaling Is Enhanced in Patients With Insufficient Coronary Collateral Artery Development and Inhibits Arteriogenesis in Mice

Stimulation of collateral artery growth in patients has been hitherto unsuccessful, despite promising experimental approaches. Circulating monocytes are involved in the growth of collateral arteries, a process also referred to as arteriogenesis. Patients show a large heterogeneity in their natural arteriogenic response on arterial obstruction. We hypothesized that circulating cell transcriptomes would provide mechanistic insights and new therapeutic strategies to stimulate arteriogenesis. Collateral flow index was measured in 45 patients with single-vessel coronary artery disease, separating collateral responders (collateral flow index, >0.21) and nonresponders (collateral flow index, ≤0.21). Isolated monocytes were stimulated with lipopolysaccharide or taken into macrophage culture for 20 hours to mimic their phenotype during arteriogenesis. Genome-wide mRNA expression analysis revealed 244 differentially expressed genes (adjusted P, <0.05) in stimulated monocytes. Interferon (IFN)-β and several IFN-related genes showed increased mRNA levels in 3 of 4 cellular phenotypes from nonresponders. Macrophage gene expression correlated with stimulated monocytes, whereas resting monocytes and progenitor cells did not display differential gene regulation. In vitro, IFN-β dose-dependently inhibited smooth muscle cell proliferation. In a murine hindlimb model, perfusion measured 7 days after femoral artery ligation showed attenuated arteriogenesis in IFN-β–treated mice compared with controls (treatment versus control: 31.5±1.2% versus 41.9±1.9% perfusion restoration, P<0.01). In conclusion, patients with differing arteriogenic response as measured with collateral flow index display differential transcriptomes of stimulated monocytes. Nonresponders show increased expression of IFN-β and its downstream targets, and IFN-β attenuates proliferation of smooth muscle cells in vitro and hampers arteriogenesis in mice. Inhibition of IFN-β signaling may serve as a novel approach for the stimulation of collateral artery growth.

Suppression of inflammatory signaling in monocytes from patients with coronary artery disease

Monocytes and T-cells play an important role in the development of atherosclerotic coronary artery disease (CAD). Transcriptome analysis of circulating mononuclear cells from carefully matched atherosclerotic and control patients will potentially provide insights into the pathophysiology of atherosclerosis and supply biomarkers for diagnostic purposes. From patients undergoing coronary angiography because of anginal symptoms, we carefully matched 18 patients with severe triple-vessel CAD to 13 control patients without angiographic signs of CAD. All patients were on statin and aspirin treatment. Elevated soluble-ICAM levels demonstrated increased vascular inflammation in atherosclerotic patients. RNA from circulating CD4+ T-cells, CD14+ monocytes, lipopolysaccharide-stimulated monocytes, and macrophages was subjected to genome-wide expression analysis. In CD14+ monocytes, few inflammatory genes were overexpressed in control patients, while atherosclerotic patients showed overexpression of a group of Krüppel-associated box - containing transcription factors involved in negative regulation of gene expression. These differences disappeared upon LPS-stimulation or differentiation towards macrophages. No consistent changes in T cell transcriptomes were detected. Large inter-individual variability prevented the use of single differentially expressed genes as biomarkers, while monocyte gene expression signature predicted patient status with an accuracy of 84%. In this comprehensive analysis of circulating cell transcriptomes in atherosclerotic CAD, cautious patient matching revealed only small differences in transcriptional activity in different mononuclear cell types. Only an indication of a negative feedback to inflammatory gene expression was detected in atherosclerotic patients. Transcriptome differences of circulating cells possibly play less of a role than hitherto thought in the individual patients susceptibility to atherosclerotic CAD, when appropriately matched for clinical symptoms and medication taken.

Folic acid supplementation normalizes the endothelial progenitor cell transcriptome of patients with type 1 diabetes: a case-control pilot study

BackgroundEndothelial progenitor cells play an important role in vascular wall repair. Patients with type 1 diabetes have reduced levels of endothelial progenitor cells of which their functional capacity is impaired. Reduced nitric oxide bioavailability and increased oxidative stress play a role in endothelial progenitor cell dysfunction in these patients. Folic acid, a B-vitamin with anti-oxidant properties, may be able to improve endothelial progenitor cell function. In this study, we investigated the gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes compared to endothelial progenitor cells from healthy subjects. Furthermore, we studied the effect of folic acid on gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes.MethodsWe used microarray analysis to investigate the gene expression profiles of endothelial progenitor cells from type 1 diabetes patients before (n = 11) and after a four week period of folic acid supplementation (n = 10) compared to the gene expression profiles of endothelial progenitor cells from healthy subjects (n = 11). The probability of genes being differentially expressed among the classes was computed using a random-variance t-test. A multivariate permutation test was used to identify genes that were differentially expressed among the two classes. Functional classification of differentially expressed genes was performed using the biological process ontology in the Gene Ontology database.ResultsType 1 diabetes significantly modulated the expression of 1591 genes compared to healthy controls. These genes were found to be involved in processes regulating development, cell communication, cell adhesion and localization. After folic acid treatment, endothelial progenitor cell gene expression profiles from diabetic patients were similar to those from healthy controls. Genes that were normalized by folic acid played a prominent role in development, such as the transcription factors ID1 and MAFF. Few oxidative-stress related genes were affected by folic acid.ConclusionFolic acid normalizes endothelial progenitor cell gene expression profiles of patients with type 1 diabetes. Signaling pathways modulated by folic acid may be potential therapeutic targets to improve endothelial progenitor cell function.

Neovascularization Capacity of Mesenchymal Stromal Cells From Critical Limb Ischemia Patients Is Equivalent to Healthy Controls

Critical limb ischemia (CLI) is often poorly treatable by conventional management and alternatives such as autologous cell therapy are increasingly investigated. Whereas previous studies showed a substantial impairment of neovascularization capacity in primary bone-marrow (BM) isolates from patients, little is known about dysfunction in patient-derived BM mesenchymal stromal cells (MSCs). In this study, we have compared CLI-MSCs to healthy controls using gene expression profiling and functional assays for differentiation, senescence and in vitro and in vivo pro-angiogenic ability. Whereas no differentially expressed genes were found and adipogenic and osteogenic differentiation did not significantly differ between groups, chondrogenic differentiation was impaired in CLI-MSCs, potentially as a consequence of increased senescence. Migration experiments showed no differences in growth factor sensitivity and secretion between CLI- and control MSCs. In a murine hind-limb ischemia model, recovery of perfusion was enhanced in MSC-treated mice compared to vehicle controls (71 ± 24% versus 44 ± 11%; P < 1 × 10(-6)). CLI-MSC- and control-MSC-treated animals showed nearly identical amounts of reperfusion (ratio CLI:Control = 0.98, 95% CI = 0.82-1.14), meeting our criteria for statistical equivalence. The neovascularization capacity of MSCs derived from CLI-patients is not compromised and equivalent to that of control MSCs, suggesting that autologous MSCs are suitable for cell therapy in CLI patients.

Polymer-based Scaffold Designs For In Situ Vascular Tissue Engineering: Controlling Recruitment and Differentiation Behavior of Endothelial Colony Forming Cells

In situ vascular tissue engineering has been proposed as a promising approach to fulfill the need for small-diameter blood vessel substitutes. The approach comprises the use of a cell-free instructive scaffold to guide and control cell recruitment, differentiation, and tissue formation at the locus of implantation. Here we review the design parameters for such scaffolds, with special emphasis on differentiation of recruited ECFCs into the different lineages that constitute the vessel wall. Next to defining the target properties of the vessel, we concentrate on the target cell source, the ECFCs, and on the environmental control of the fate of these cells within the scaffold. The prospects of the approach are discussed in the light of current technical and biological hurdles.

Galectin-2 expression is dependent on the rs7291467 polymorphism and acts as an inhibitor of arteriogenesis

AIMS In patients with obstructive coronary artery disease (CAD), the growth of collateral arteries, i.e. arteriogenesis, can preserve myocardial tissue perfusion and function. Monocytes modulate this process, supplying locally the necessary growth factors and degrading enzymes. Knowledge on factors involved in human arteriogenesis is scarce. Thus, the aim of the present study is to identify targets in monocytes that are critical for arteriogenesis in patients with CAD. METHODS AND RESULTS A total of 50 patients with a chronic total coronary occlusion were dichotomized according to their collateral flow index. From each patient, RNA was isolated from unstimulated peripheral blood monocytes, monocytes stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, and from macrophages. Increased mRNA expression of galectin-2 was found in three out of four monocytic cell types of patients with a low capacity of the collateral circulation (P= 0.03 for unstimulated monocytes; P= 0.02 for LPS-stimulated monocytes; P= 0.20 for IL-4-stimulated monocytes; P= 0.02 for macrophages). Additionally, galectin-2 mRNA expression was significantly associated with the rs7291467 polymorphism in LGALS2 encoding galectin-2 in all four monocytic cell types. Patient with the rs7291467 CC genotype displayed highest galectin-2 expression, and also tended to have a lower arteriogenic response. To evaluate the effect of galectin-2 on arteriogenesis in vivo, we used a murine hindlimb model. Treatment with galectin-2 markedly impaired the perfusion restoration at Day 7. CONCLUSION Collectively, these results identify galectin-2 as a novel inhibitor of arteriogenesis. Modulation of galectin-2 may constitute a new therapeutic strategy for the stimulation of arteriogenesis in patients with CAD.