Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition.

Background: The deSUMOylase sentrin-specific isopeptidase 2 (SENP2) plays a crucial role in atheroprotection. However, the phosphorylation of SENP2 at T368 under disturbed flow (D-flow) conditions hinders its nuclear function and promotes endothelial cell (EC) activation. SUMOylation has been implicated in D-flow-induced endothelial-to-mesenchymal transition (endoMT), but the precise role of SENP2 in counteracting this process remains unclear. Method: We developed a phospho-specific SENP2 S344 antibody and generated knock-in (KI) mice with a phospho-site mutation of SENP2 S344A using CRISPR/Cas9 technology. We then investigated the effects of SENP2 S344 phosphorylation under two distinct flow patterns and during hypercholesteremia (HC)-mediated EC activation. Result: Our findings demonstrate that laminar flow (L-flow) induces phosphorylation of SENP2 at S344 through the activation of checkpoint kinase 1 (CHK1), leading to the inhibition of ERK5 and p53 SUMOylation and subsequent suppression of EC activation. We observed a significant increase in lipid-laden lesions in both the aortic arch (under D-flow) and descending aorta (under L-flow) of female hypercholesterolemic SENP2 S344A KI mice. In male hypercholesterolemic SENP2 S344A KI mice, larger lipid-laden lesions were only observed in the aortic arch area, suggesting a weaker HC-mediated atherogenesis in male mice compared to females. Ionizing radiation (IR) reduced CHK1 expression and SENP2 S344 phosphorylation, attenuating the pro-atherosclerotic effects observed in female SENP2 S344A KI mice after bone marrow transplantation (BMT), particularly in L-flow areas. The phospho-site mutation SENP2 S344A upregulates processes associated with EC activation, including inflammation, migration, and proliferation. Additionally, fibrotic changes and up-regulated expression of EC marker genes were observed. Apoptosis was augmented in ECs derived from the lungs of SENP2 S344A KI mice, primarily through the inhibition of ERK5-mediated expression of DNA damage-induced apoptosis suppressor (DDIAS). Summary: In this study, we have revealed a novel mechanism underlying the suppressive effects of L-flow on EC inflammation, migration, proliferation, apoptosis, and fibrotic changes through promoting CHK1-induced SENP2 S344 phosphorylation. The phospho-site mutation SENP2 S344A responds to L-flow through a distinct mechanism, which involves the upregulation of both mesenchymal and EC marker genes.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Free Cholesterol Bioavailability and Atherosclerosis.

PURPOSE OF REVIEW: As both a cholesterol acceptor and carrier in the reverse cholesterol transport (RCT) pathway, high-density lipoprotein (HDL) is putatively atheroprotective. However, current pharmacological therapies to increase plasma HDL cholesterol (HDL-c) concentration have paradoxically failed to prevent or reduce atherosclerosis and cardiovascular disease (CVD). Given that free cholesterol (FC) transfer between surfaces of lipoproteins and cells is reversible, excess plasma FC can be transferred to the cells of peripheral tissue sites resulting in atherosclerosis. Here, we summarize potential mechanisms contributing to this paradox and highlight the role of excess free cholesterol (FC) bioavailability in atherosclerosis vs. atheroprotection. RECENT FINDINGS: Recent findings have established a complex relationship between HDL-c concentration and atherosclerosis. Systemic scavenger receptor class B type 1 (SR-B1) knock out (KO) mice exhibit with increased diet-induced atherosclerosis despite having an elevated plasma HDL-c concentration compared to wild type (WT) mice. The greater bioavailability of HDL-FC in SR-B1 vs. WT mice is associated with a higher FC content in multiple cell types and tissue sites. These results suggest that dysfunctional HDL with high FC bioavailability is atheroprone despite high HDL-c concentration. Past oversimplification of HDL-c involvement in cholesterol transport has led to the failures in HDL targeted therapy. Evidence suggests that FC-mediated functionality of HDL is of higher importance than its quantity; as a result, deciphering the regulatory mechanisms by which HDL-FC bioavailability can induce atherosclerosis can have far-reaching clinical implications.

Senescence-Associated Secretory Phenotype as a Hinge Between Cardiovascular Diseases and Cancer.

Overlapping risks for cancer and cardiovascular diseases (CVD), the two leading causes of mortality worldwide, suggest a shared biology between these diseases. The role of senescence in the development of cancer and CVD has been established. However, its role as the intersection between these diseases remains unclear. Senescence was originally characterized by an irreversible cell cycle arrest after a high number of divisions, namely replicative senescence (RS). However, it is becoming clear that senescence can also be instigated by cellular stress, so-called stress-induced premature senescence (SIPS). Telomere shortening is a hallmark of RS. The contribution of telomere DNA damage and subsequent DNA damage response/repair to SIPS has also been suggested. Although cellular senescence can mediate cell cycle arrest, senescent cells can also remain metabolically active and secrete cytokines, chemokines, growth factors, and reactive oxygen species (ROS), so-called senescence-associated secretory phenotype (SASP). The involvement of SASP in both cancer and CVD has been established. In patients with cancer or CVD, SASP is induced by various stressors including cancer treatments, pro-inflammatory cytokines, and ROS. Therefore, SASP can be the intersection between cancer and CVD. Importantly, the conventional concept of senescence as the mediator of cell cycle arrest has been challenged, as it was recently reported that chemotherapy-induced senescence can reprogram senescent cancer cells to acquire "stemness" (SAS: senescence-associated stemness). SAS allows senescent cancer cells to escape cell cycle arrest with strongly enhanced clonogenic growth capacity. SAS supports senescent cells to promote both cancer and CVD, particularly in highly stressful conditions such as cancer treatments, myocardial infarction, and heart failure. As therapeutic advances have increased overlapping risk factors for cancer and CVD, to further understand their interaction may provide better prevention, earlier detection, and safer treatment. Thus, it is critical to study the mechanisms by which these senescence pathways (SAS/SASP) are induced and regulated in both cancer and CVD.

Disturbed flow-induced FAK K152 SUMOylation initiates the formation of pro-inflammation positive feedback loop by inducing reactive oxygen species production in endothelial cells.

Focal adhesion kinase (FAK) activation plays a crucial role in vascular diseases. In endothelial cells, FAK activation is involved in the activation of pro-inflammatory signaling and the progression of atherosclerosis. Disturbed flow (D-flow) induces endothelial activation and senescence, but the exact role of FAK in D-flow-induced endothelial activation and senescence remains unclear. The objective of this study is to investigate the role of FAK SUMOylation in D-flow-induced endothelial activation and senescence. The results showed that D-flow induced reactive oxygen species (ROS) production via NADPH oxidase activation and activated a redox-sensitive kinase p90RSK, leading to FAK activation by upregulating FAK K152 SUMOylation and the subsequent Vav2 phosphorylation, which in turn formed a positive feedback loop by upregulating ROS production. This feedback loop played a crucial role in regulating endothelial activation and senescence. D-flow-induced endothelial activation and senescence were significantly inhibited by mutating a FAK SUMOylation site lysine152 to arginine. Collectively, we concluded that FAK K152 SUMOylation plays a key role in D-flow-induced endothelial activation and senescence by forming a positive feedback loop through ROS production.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells.

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer. Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50-70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11. Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2. Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Corrigendum: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway.

[This corrects the article DOI: 10.3389/fcvm.2020.542485.].

p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway.

Previously, we reported that post-translational modifications (PTMs) of MAGI1, including S741 phosphorylation and K931 de-SUMOylation, both of which are regulated by p90RSK activation, lead to endothelial cell (EC) activation. However, roles for p90RSK and MAGI1-PTMs in regulating EC permeability remain unclear despite MAGI1 being a junctional molecule. Here, we show that thrombin (Thb)-induced EC permeability, detected by the electric cell-substrate impedance sensing (ECIS) based system, was decreased by overexpression of dominant negative p90RSK or a MAGI1-S741A phosphorylation mutant, but was accelerated by overexpression of p90RSK, siRNA-mediated knockdown of magi1, or the MAGI1-K931R SUMOylation mutant. MAGI1 depletion also increased the mRNA and protein expression of the large tumor suppressor kinases 1 and 2 (LATS1/2), which inhibited YAP/TAZ activity and increased EC permeability. Because the endothelial barrier is a critical mediator of tumor hypoxia, we also evaluated the role of p90RSK activation in tumor vessel leakiness by using a relatively low dose of the p90RSK specific inhibitor, FMK-MEA. FMK-MEA significantly inhibited tumor vessel leakiness at a dose that does not affect morphology and growth of tumor vessels in vivo. These results provide novel insights into crucial roles for p90RSK-mediated MAGI1 PTMs and the Hippo pathway in EC permeability, as well as p90RSK activation in tumor vessel leakiness.

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1-/+ mice inhibited d-flow-induced atherogenesis. In sum, EC activation and ER stress-mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

Ponatinib Activates an Inflammatory Response in Endothelial Cells via ERK5 SUMOylation.

Ponatinib is a multi-targeted third generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML) patients harboring the Abelson (Abl)-breakpoint cluster region (Bcr) T315I mutation. In spite of having superb clinical efficacy, ponatinib triggers severe vascular adverse events (VAEs) that significantly limit its therapeutic potential. On vascular endothelial cells (ECs), ponatinib promotes EC dysfunction and apoptosis, and inhibits angiogenesis. Furthermore, ponatinib-mediated anti-angiogenic effect has been suggested to play a partial role in systemic and pulmonary hypertension via inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Even though ponatinib-associated VAEs are well documented, their etiology remains largely unknown, making it difficult to efficiently counteract treatment-related adversities. Therefore, a better understanding of the mechanisms by which ponatinib mediates VAEs is critical. In cultured human aortic ECs (HAECs) treated with ponatinib, we found an increase in nuclear factor NF-kB/p65 phosphorylation and NF-kB activity, inflammatory gene expression, cell permeability, and cell apoptosis. Mechanistically, ponatinib abolished extracellular signal-regulated kinase 5 (ERK5) transcriptional activity even under activation by its upstream kinase mitogen-activated protein kinase kinase 5α (CA-MEK5α). Ponatinib also diminished expression of ERK5 responsive genes such as Krüppel-like Factor 2/4 (klf2/4) and eNOS. Because ERK5 SUMOylation counteracts its transcriptional activity, we examined the effect of ponatinib on ERK5 SUMOylation, and found that ERK5 SUMOylation is increased by ponatinib. We also found that ponatibib-mediated increased inflammatory gene expression and decreased anti-inflammatory gene expression were reversed when ERK5 SUMOylation was inhibited endogenously or exogenously. Overall, we propose a novel mechanism by which ponatinib up-regulates endothelial ERK5 SUMOylation and shifts ECs to an inflammatory phenotype, disrupting vascular homeostasis.

Olive Oil Polyphenols Differentially Inhibit Smooth Muscle Cell Proliferation through a G1/S Cell Cycle Block Regulated by ERK1/2.

We hypothesized that polyphenols contained in olive oil play a role in reducing the risk of atherosclerosis. The aim of this study was to determine if the polyphenols in olive oil, oleuropein (Ole), hydroxytyrosol (HT), and tyrosol (Tyr) could inhibit smooth muscle cell (SMC) proliferation through its influence on cell cycle regulation. Bovine vascular SMC were cultured in the presence of Ole, HT, or Tyr at concentration of 1, 10, or 100 µmol/L. On days 1, 3, and 5, numbers of cells were counted. Cell cycle analysis was performed by flow cytometry on day 1 after SMC were stained with propidium iodide. Cell populations grown in the presence of Ole or HT at 100 µmol/L concentration were significantly inhibited after 5 days of exposure. Tyr had a similar tendency but it did not attain significance. Cell cycle analysis revealed that 66% of cells were in G1 phase in Ole group, compared with 48% in control group. To examine the cell cycle block between G1 and S phases, we performed Western blotting and found that ERK1/2 activation was inhibited by Ole or HT. We conclude that olive oil polyphenols could inhibit SMC proliferation through a cell cycle block between G1 and S phases which may be regulated by ERK1/2. These results demonstrate a mechanism by which olive oil consumption may be atheroprotective by inhibiting SMC proliferation.

Varying effects of hemodynamic forces on tissue factor RNA expression in human endothelial cells.

BACKGROUND: Atherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli. MATERIALS AND METHODS: TF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS). RESULTS: Mechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LF+Th for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PF+Th (14.8 ± 2.4) and TFF+Th (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th. CONCLUSIONS: While uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.

Laminar shear, but not orbital shear, has a synergistic effect with thrombin stimulation on tissue factor expression in human umbilical vein endothelial cells.

INTRODUCTION: High levels of tissue factor (TF) have been associated with atherosclerotic plaques. The specific pathways linked to TF expression in endothelial cells (ECs) have not been well defined. This study compared TF expression in human umbilical vein ECs (HUVECs) exposed to laminar shear stress (LSS) using a parallel flow chamber and to orbital shear stress (OSS) using an orbital shaker. We also compared the effects of thrombin (TH) stimulation of ECs exposed to different shear forces on the expression of TF and investigated the role that second messengers, p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2), had in the EC response. METHODS: HUVECs were subjected to 2, 4, or 6 hours of LSS or OSS in the presence or absence of 4 U/mL of TH. Western blot analysis of ERK1/2 and p38 activation and polymerase chain reaction analysis of TF in the presence of inhibitors to these second messengers was performed in HUVECs subjected to OSS or LSS in the presence or absence of TH. RESULTS: TF expression was increased and peaked at 2 hours in all HUVECs exposed to LSS or TH. Stimulation of static HUVECs with TH resulted in an increase in TF expression of 5.68 ± 1.58-, 3.80 ± 1.21-, and 2.54 ± 0.38-fold at 2, 4, and 6 hours, respectively (n = 6 experiments). In the absence of TH, HUVECs exposed to LSS demonstrated a 9.51 ± 0.62-, 7.31 ± 1.43-, and 4.39 ± 1.32-fold increase in TF expression at 2, 4, and 6 hours, respectively (n = 6 experiments). TF was increased significantly more when exposed to LSS in the presence of TH (18.85 ± 1.43-, 15.05 ± 0.95-, and 8.91 ± 1.06-fold increases at 2, 4, and 6 hours, respectively [n = 6 experiments], P < .01). Between-group analysis showed a significant difference between groups (P < .001). OSS did not significantly increase TF expression in the presence or absence of TH. ERK1/2 and p38 activation was increased in LSS and LSS + TH but not in OSS or OSS + TH (n = 3 experiments). CONCLUSION: LSS and TH independently increased TF expression, but OSS did not. LSS + TH stimulation showed a synergistic effect, which suggests that these mechanical and chemical stimuli work through different pathways or that an intracellular interaction between TH and LSS may be present that does not occur in OSS.

Pulsatile to-fro flow induces greater and sustained expression of tissue factor RNA in HUVEC than unidirectional laminar flow.

Tissue factor (TF) is expressed in atherosclerotic lesions. Since mechanical forces influence endothelial cell (EC) function and are thought to account for the unique distribution of atherosclerosis in areas exposed to disturbed flow, we hypothesized that disturbed to-fro flow (TFF) and unidirectional pulsatile forward flow (PFF) would have different effects on TF expression in EC. TF RNA expression in HUVEC exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin was determined. TFF induced a significantly higher TF expression than PFF that was sustained for 8 h. Combination of mechanical and chemical stimuli induced significantly higher TF expression than only mechanical stresses, and this effect was synergistic in both TFF and PFF. The MAPK p38 inhibitor SB-203580 significantly inhibited TF expression induced by mechanical and chemical stimulations, but the MEK inhibitor PD-98059 did not inhibit TF induced by TFF. Immunoblotting revealed that ERK1/2 phosphorylation induced by TFF was sustained for 120 min, whereas that induced by PFF was not. We conclude that disturbed flow induced greater and sustained amplification of TF expression, and this synergistic effect may be regulated by p38 MAPK and ERK1/2. These results provide added insight into the mechanism of atherosclerosis in areas of disturbed flow.