Reciprocal regulation of endothelial-mesenchymal transition by MAPK7 and EZH2 in intimal hyperplasia and coronary artery disease.

Endothelial-mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFß1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

In Pulmonary Arterial Hypertension, Reduced BMPR2 Promotes Endothelial-to-Mesenchymal Transition via HMGA1 and Its Target Slug.

BACKGROUND: We previously reported high-throughput RNA sequencing analyses that identified heightened expression of the chromatin architectural factor High Mobility Group AT-hook 1 (HMGA1) in pulmonary arterial endothelial cells (PAECs) from patients who had idiopathic pulmonary arterial hypertension (PAH) in comparison with controls. Because HMGA1 promotes epithelial-to-mesenchymal transition in cancer, we hypothesized that increased HMGA1 could induce transition of PAECs to a smooth muscle (SM)-like mesenchymal phenotype (endothelial-to-mesenchymal transition), explaining both dysregulation of PAEC function and possible cellular contribution to the occlusive remodeling that characterizes advanced idiopathic PAH. METHODS AND RESULTS: We documented increased HMGA1 in PAECs cultured from idiopathic PAH versus donor control lungs. Confocal microscopy of lung explants localized the increase in HMGA1 consistently to pulmonary arterial endothelium, and identified many cells double-positive for HMGA1 and SM22α in occlusive and plexogenic lesions. Because decreased expression and function of bone morphogenetic protein receptor 2 (BMPR2) is observed in PAH, we reduced BMPR2 by small interfering RNA in control PAECs and documented an increase in HMGA1 protein. Consistent with transition of PAECs by HMGA1, we detected reduced platelet endothelial cell adhesion molecule 1 (CD31) and increased endothelial-to-mesenchymal transition markers, αSM actin, SM22α, calponin, phospho-vimentin, and Slug. The transition was associated with spindle SM-like morphology, and the increase in αSM actin was largely reversed by joint knockdown of BMPR2 and HMGA1 or Slug. Pulmonary endothelial cells from mice with endothelial cell-specific loss of Bmpr2 showed similar gene and protein changes. CONCLUSIONS: Increased HMGA1 in PAECs resulting from dysfunctional BMPR2 signaling can transition endothelium to SM-like cells associated with PAH.

Endothelial Plasticity: Shifting Phenotypes through Force Feedback.

The endothelial lining of the vasculature is exposed to a large variety of biochemical and hemodynamic stimuli with different gradients throughout the vascular network. Adequate adaptation requires endothelial cells to be highly plastic, which is reflected by the remarkable heterogeneity of endothelial cells in tissues and organs. Hemodynamic forces such as fluid shear stress and cyclic strain are strong modulators of the endothelial phenotype and function. Although endothelial plasticity is essential during development and adult physiology, proatherogenic stimuli can induce adverse plasticity which contributes to disease. Endothelial-to-mesenchymal transition (EndMT), the hallmark of endothelial plasticity, was long thought to be restricted to embryonic development but has emerged as a pathologic process in a plethora of diseases. In this perspective we argue how shear stress and cyclic strain can modulate EndMT and discuss how this is reflected in atherosclerosis and pulmonary arterial hypertension.

FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-ß signaling.

Endothelial-to-mesenchymal transition (EndMT) is characterized by the loss of endothelial cell markers and functions, and coincides with de novo expression of mesenchymal markers. EndMT is induced by TGFß1 and changes endothelial microRNA expression. We found that miR-20a is decreased during EndMT, and that ectopic expression of miR-20a inhibits EndMT induction. TGFß1 induces cellular hypertrophy in human umbilical vein endothelial cells and abrogates VE-cadherin expression, reduces endothelial sprouting capacity and induces the expression of the mesenchymal marker SM22α (also known as TAGLN). We identified ALK5 (also known as TGFBR1), TGFBR2 and SARA (also known as ZFYVE9) as direct miR-20a targets. Expression of miR-20a mimics abrogate the endothelial responsiveness to TGFß1, by decreasing ALK5, TGFBR2 and SARA, and inhibit EndMT, as indicated by the maintenance of VE-cadherin expression, the ability of the cells to sprout and the absence of SM22α expression. FGF2 increases miR-20a expression and inhibits EndMT in TGFß1-stimulated endothelial cells. In summary, FGF2 controls endothelial TGFß1 signaling by regulating ALK5, TGFBR2 and SARA expression through miR-20a. Loss of FGF2 signaling combined with a TGFß1 challenge reduces miR-20a levels and increases endothelial responsiveness to TGFß1 through elevated receptor complex levels and activation of Smad2 and Smad3, which culminates in EndMT.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS: Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS: In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION: Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

AIMS: The MEK5/Erk5 pathway mediates beneficial effects of laminar flow, a major physiological factor preventing vascular dysfunction. Forced Erk5 activation induces a protective phenotype in endothelial cell (EC) that is associated with a dramatically decreased migration capacity of those cells. Transcriptional profiling identified the Krüppel-like transcription factors KLF2 and KLF4 as central mediators of Erk5-dependent gene expression. However, their downstream role regarding migration is unclear and relevant secondary effectors remain elusive. Here, we further investigated the mechanism underlying Erk5-dependent migration arrest in ECs. METHODS AND RESULTS: Our experiments reveal KLF2-dependent loss of the pro-migratory Rac/Cdc42 mediator, p21-activated kinase 1 (PAK1), as an important mechanism of Erk5-induced migration inhibition. We show that endothelial Erk5 activation by expression of a constitutively active MEK5 mutant, by statin treatment, or by application of laminar shear stress strongly decreased PAK1 mRNA and protein expression. Knockdown of KLF2 but not of KLF4 prevented Erk5-mediated PAK1 mRNA inhibition, revealing KLF2 as a novel PAK1 repressor in ECs. Importantly, both PAK1 re-expression and KLF2 knockdown restored the migration capacity of Erk5-activated ECs underscoring their functional relevance downstream of Erk5. CONCLUSION: Our data provide first evidence for existence of a previously unknown Erk5/KLF2/PAK1 axis, which may limit undesired cell migration in unperturbed endothelium and lower its sensitivity for migratory cues that promote vascular diseases including atherosclerosis.

The flow dependency of Tie2 expression in endotoxemia.

RATIONALE: Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction. OBJECTIVE: To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge. METHODS AND RESULTS: Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1ß. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge. CONCLUSIONS: Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1ß challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

IL-1ß and TGFß2 synergistically induce endothelial to mesenchymal transition in an NFκB-dependent manner.

Endothelial to mesenchymal transition (EndMT) contributes to fibrotic diseases. The main inducer of EndMT is TGFß signaling. TGFß2 is the dominant isoform in the physiological embryonic EndMT, but its role in the pathological EndMT in the context of inflammatory co-stimulation is not known. The aim of this study was to investigate TGFß2-induced EndMT in the context of inflammatory IL-1ß signaling. Co-stimulation with IL-1ß and TGFß2, but not TGFß1, caused synergistic induction of EndMT. Also, TGFß2 was the only TGFß isoform that was progressively upregulated during EndMT. External IL-1ß stimulation was dispensable once EndMT was induced. The inflammatory transcription factor NFκB was upregulated in an additive manner by IL-1ß and TGFß2 co-stimulation. Co-stimulation also led to the nuclear translocation of NFκB which was sustained over long-term treatment. Activation of NFκB was indispensable for the co-induction of EndMT. Our data suggest that the microenvironment at the verge between inflammation (IL-1ß) and tissue remodeling (TGFß2) can strongly promote the process of EndMT. Therefore our findings provide new insights into the mechanisms of pathological EndMT.

Cellular plasticity: the good, the bad, and the ugly? Microenvironmental influences on progenitor cell therapy.

Progenitor cell based therapies have emerged for the treatment of ischemic cardiovascular diseases where there is insufficient endogenous repair. However, clinical success has been limited, which challenges the original premise that transplanted progenitor cells would orchestrate repair. In this review, we discuss the basics of endothelial progenitor cell therapy and describe how microenvironmental changes (i.e., trophic and mechano-structural factors) in the damaged myocardium influence progenitor cell plasticity and hamper beneficial therapeutic outcome. Further understanding of these microenvironmental clues will enable optimization of cell therapy at all levels. We discuss current concepts and provide future perspectives for the enhancement of progenitor cell therapy, and merge these advances into a combined approach for ischemic tissue repair.

Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny.

AIMS: Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGFbeta1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains circulating endothelial progenitor cells of which the endothelial colony-forming cells (ECFCs) harbour stem cell-like properties. Given the plasticity between endothelial and mesenchymal lineages and the stem cell-like properties of ECFCs, we hypothesized that ECFCs can give rise to smooth muscle-like progeny. METHODS AND RESULTS: ECFCs were stimulated with TGFbeta1, after which TGFbeta signalling cascades and their downstream effects were investigated. Indeed, EndMT of ECFCs resulted in smooth muscle-like progeniture. TGFbeta1-driven EndMT is mediated by ALK5 kinase activity, increased downstream Smad2 signalling, and reduced protein levels of inhibitor of DNA-binding protein 3. ECFCs lost expression of endothelial markers and endothelial anti-thrombogenic function. Simultaneously, mesenchymal marker expression was gained, cytoskeletal rearrangements occurred, and cells acquired a contractile phenotype. Transdifferentiated ECFCs were phenotypically stable and self-sustaining and, importantly, showed fibroblast growth factor-2 and angiopoietin-1-mediated pro-angiogenic paracrine properties. CONCLUSION: Our study is the first to demonstrate that ECFCs can give rise to smooth muscle-like progeny, with potential therapeutic benefits. These findings further illustrate that ECFCs are highly plastic, which by itself has implications for therapeutical use.

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices.

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.

Generating new blood flow: integrating developmental biology and tissue engineering.

Vascular tissue engineering aims to restore blood flow by seeding artificial tubular scaffolds with endothelial and smooth muscle cells, thus creating bioartificial blood vessels. Herein, the progenitors of smooth muscle and endothelial cells hold great promise because they efficiently differentiate and harbor longevity. In this review, we describe a novel tissue engineering approach that uses current insights from developmental biology, that is, progenitor cell plasticity, and the latest advances in biomaterial design. We focus specifically on developmental processes that regulate progenitor cell (trans)differentiation and offer a platform for the integration of these molecular clues into biomaterial design. We propose a novel engineering paradigm for the creation of a small-diameter blood vessel wherein progenitor cell differentiation and tissue organization are instructed by the biomaterial solely. With this review, we emphasize the power of integrating developmental biology and material science for vascular tissue engineering.

Reduced number and impaired function of circulating progenitor cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34-CD133 double-positive cells (P < 0.001) as well as the CFU capacity (P = 0.048) was reduced in SLE patients. Migratory activity on tumor necrosis factor-alpha tended to be reduced in patient CPCs (P = 0.08). Migration on vascular endothelial growth factor showed no significant differences, nor were differences observed in the potential to form clusters on Matrigel. The expression of caspase 8L was reduced at the transcriptional level (P = 0.049) and strongly increased at the protein level after culture (P = 0.003). We conclude that CPC numbers are reduced in SLE patients and functionality is partly impaired. We suggest these findings reflect increased susceptibility to apoptosis of CPCs from SLE patients.