MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.     

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs’ proliferation and migration. Over-expression of Bcl-2 increased HAECs’ tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation.Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

Secreted frizzled‐related protein 5 protects against oxidative stress‐induced apoptosis in human aortic endothelial cells via downregulation of Bax

This study was undertaken to determine the role of secreted frizzled‐related protein 5 (SFRP5) in endothelial oxidative injury. Human aortic endothelial cells (HAECs) were exposed to different oxidative stimuli and examined for SFRP5 expression. The effects of SFRP5 overexpression and knockdown on cell viability, apoptosis, and reactive oxygen species production were measured. HAECs treated with angiotensin (Ang) II (1 μM) or oxidized low‐density lipoprotein (oxLDL) (150 μg/mL) showed a significant increase in SFRP5 expression. Overexpression of SFRP5 significantly attenuated the viability suppression and apoptosis induction by Ang II and oxLDL, whereas the knockdown of SFRP5 exerted opposite effects. Overexpression of SFRP5 prevented ROS formation and β‐catenin activation and reduced Bax expression. Co‐expression of Bax significantly reversed the anti‐apoptotic effect of SFRP5 overexpression, whereas knockdown of Bax restrained Ang II‐ and oxLDL‐induced apoptosis in HAECs. Taken together, SFRP5 confers protection against oxidative stress‐induced apoptosis through inhibition of β‐catenin activation and downregulation of Bax.     

Erythropoietin attenuated high glucose-induced apoptosis in cultured human aortic endothelial cells

High glucose-induced apoptosis in vascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes. Here, we show that erythropoietin attenuates high glucose-induced apoptosis in cultured human aortic endothelial cells (HAECs). Exposure of HAECs to high glucose level for 72h significantly increased the number of apoptotic cells compared with normal glucose level, as evaluated by TUNEL assay. Simultaneous addition of erythropoietin (100 U/ml) significantly attenuated high glucose-induced apoptosis. In parallel, exposure to high glucose level induced caspase-3 activation and erythropoietin also prevented it. Erythropoietin stimulated Akt phosphorylation in a dose-dependent manner (1-100 U/ml). PI3 kinase inhibitor, wortmannin or LY294002 eliminated erythropoietin's inhibitory effect on caspase-3 activity. In conclusion, erythropoietin may attenuate high glucose-induced endothelial cell apoptosis via PI-3 kinase pathway. Replacing therapy with erythropoietin is often used for correction of renal anemia, but may have potential in preventing atherosclerosis in diabetic patients with end-stage renal failure.     

Induction of stearoyl-CoA desaturase protects human arterial endothelial cells against lipotoxicity

Endothelial lipotoxicity has been implicated in the pathogenesis of multiple stages of cardiovascular disease from early endothelial dysfunction to manifest atherosclerosis and its complications. Saturated free fatty acids are the major inducers of endothelial cell apoptosis and inflammatory cytokines. In humans, the enzyme human stearoyl-CoA desaturase-1 (hSCD-1) is the limiting step of the desaturation of saturated to monounsaturated fatty acids. Since we could demonstrate the expression of SCD-1 in primary human arterial endothelial cells (HAECs), we aimed to prove a beneficial role of upregulated hSCD-1 expression. In contrast to other cells that are less susceptible to lipotoxicity, hSCD-1 was not upregulated in HAECs upon palmitate treatment. Following that, we could show that upregulation of hSCD-1 using the LXR activator TO-901317 in HAECs protects the cells against palmitate-induced lipotoxicity, cell apoptosis, and expression of inflammatory cytokines IL-6 and IL-8. Increased hSCD-1 activity was determined as increased C16:1/16:0 ratio and enhanced triglyceride storage in palmitate treated cells. The beneficial effect was clearly attributed to enhanced hSCD-1 activity. Overexpression of hSCD-1 blocked palmitate-induced cytotoxicity, and knockdown of hSCD-1 using siRNA abolished the protective effect of TO-901317 in HEK-293 cells. Additionally, inhibition of hSCD-1 with 10/12 CLA blocked the effect of TO-901317 on palmitate-induced lipotoxicity, cell apoptosis, and inflammatory cytokine induction in HAECs. We conclude that upregulation of hSCD-1 leads to a desaturation of saturated fatty acids and facilitates their esterification and storage, thereby preventing downstream effects of lipotoxicity in HAECs. These findings add a novel aspect to the atheroprotective actions of LXR activators in cardiovascular disease.     

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H₂S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H₂S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs‐926) with NaHS (a H₂S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H₂S had little effect on eNOS protein expression in ECs. L‐cysteine, a precursor of H₂S, stimulated NO production whereas blockage of the activity of H₂S‐generating enzyme, cystathionine gamma‐lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3‐K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H₂S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS‐specific siRNA or nitro‐L‐arginine methyl ester (L‐NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H₂S on ECs. Our results suggest that H₂S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt‐dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H₂S‐induced angiogenesis.     

Atherosclerosis-associated endothelial cell apoptosis by miRNA let7-b-mediated downregulation of HAS-2

MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various types of human diseases including atherosclerosis. Increasing numbers of miRNAs have been identified to be important regulators in the progression of atherosclerosis by regulating gene expression. However, functional miRNAs and the underlying mechanisms involved in atherosclerosis need fully elucidation. In the present study, the function of miRNA let-7b was investigated in human aortic endothelial cells (HAECs). The results showed that downregulation of let-7b in the high-fat diet mice and HAECs was inversely correlated with the expression level of HAS-2. upregulation of let-7b significantly reduced apoptosis of HAECs. The results also revealed that HAS-2 was a target gene of let-7b and HAS-2 reduction reversed the antiapoptotic effect of let-7b through regulation of the P13K/Akt pathway. These results together suggest the potential of regulating the let-7b expression and endothelial apoptosis against development and progression of atherosclerosis.     

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.     

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.     

C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products

Objective

C-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of atherosclerosis. CRP can regulate receptor for advanced glycation end-products (RAGE) expression in endothelial progenitor cells (EPCs). Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. It is believed that decreased eNOS bioactivity occurs early in atherogenesis. Therefore, we tested the hypothesis that CRP can alter eNOS expression and promote apoptosis in EPCs through RAGE.

Methods and results

EPCs, isolated from bone marrow, were cultured in the presence or absence of LPS-free CRP (5, 10, 15, 20, and50 μg/ml). RAGE protein expression and siRNA were measured by flow cytometric analysis. PCR was used to detect eNOS mRNA expression. eNOS protein expression was measured by Western blot analysis. A spectrophotometer was used to assess eNOS activity. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure adhesiveness. A MTT assay was used to determine proliferation. Apoptosis was evaluated by annexin V immunostaining and TUNEL staining. Co-culturing with CRP caused a significant down-regulation of eNOS expression, inhibited the proliferation, migration, and adhesion of EPCs, and induced EPC apoptosis. In addition, these effects were attenuated during RAGE protein expression blockade by siRNA.

Conclusions

CRP, at concentrations known to predict cardiovascular event, directly quenches the expression of eNOS and diminishes NO production, and may serve to impair EPC function and promote EPC apoptosis through RAGE. These data further support a direct role of CRP in the development and/or progression of atherosclerosis and indicate a new pathophysiologic mechanism of disturbed vascular adaptation in atherosclerosis.     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Sophocarpine exert protective effect against ox-LDL-induced endothelial damage via regulating NF-κB signaling pathway

ABSTRACT

Oxidized low-density lipoprotein (ox-LDL) was known to induce endothelial cell injury to the progression of atherosclerosis (AS). Sophocarpine (SPC), a compound of sophora alkaloids isolated from the plant Sophora alopecuroides, has been shown to exhibit various pharmacological activities. This study was designed to investigate the protective effect of SPC on ox-LDL-induced endothelial cells and explored its underlying mechanism. Our results show that SPC pre-incubation ameliorated ox-LDL-mediated HAECs cytotoxicity, DNA fragmentation, and apoptosis in a dose-dependent manner. Moreover, SPC significantly downregulated the mRNA or protein expression level of pro-inflammatory mediators (TGF-β, IL-6, IL-1β, TNF-α) and pro-inflammatory vascular adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Mechanistically, SPC pre-treatment downregulated IκBα expression and inhibited translocation of NF-κB in ox-LDL-mediated HAECs, overexpression of NF-κB p65 counteracted the cytoprotective and anti-apoptotic effect of SPC, suggesting that its action is dependent on NF-κB signaling pathway. Collectively, SPC suppresses ox-LDL-induced HAECs injury by inhibiting the NF-κB signaling pathway.     

Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.     

Heparin Cofactor II,a Serine Protease Inhibitor,Promotes Angiogenesis via Activation of the AMP-activated Protein Kinase-Endothelial Nitric-oxide Synthase Signaling Pathway

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII^+/−) mice and male littermate wild-type (HCII^+/+) mice at the age of 12–16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII^+/− mice compared with that in HCII^+/+ mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII^+/− mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII^+/− mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.     

Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation

Calpains are a family of calcium-dependent proteases. Two isoforms, calpain 1 and 2, have been implicated in angiogenesis and endothelial cell adhesion and migration. Calpains regulate the function of eNOS;however, the relation of calpains and eNOS to lymphangiogenesisis still unclear. In the present study, we evaluated the role of calpain and eNOS in the formation of cords by lymphatic endothelial cells on Matrigel. Human lymphatic microvascular dermal-derived endothelial cells were transfected with siRNA against calpain 1 or 2. Calpain 2 knockdown, but not calpain 1 knockdown, significantly reduced cord formation, adhesion, and migration on Matrigel. These decreases correlated with a reduction in eNOS, and phosphorylated eNOS and Hsp90 levels, as assayed by immunoprecipitation and western blotting. In contrast, the knockdown of calpain 1, but not calpain 2,increased cell adhesion, enhanced migration, and stabilized late-stage cord formation by increasing cord length compared to the control. These differences correlated with an increase in the level of phosphorylated eNOS. The results indicated that the functions of calpains and eNOS are important for cord formation by lymphatic endothelial cells. For the first time, we have found different functions of calpain 1 and 2. Calpain 1 is involved in the degradation of eNOS and Hsp90 and the phosphorylation of eNOS,while calpain 2 regulates eNOS phosphorylation during cord formation by lymphatic endothelial cells on Matrigel.     

A protective role of ciglitazone in ox‐LDL‐induced rat microvascular endothelial cells via modulating PPARγ‐dependent AMPK/eNOS pathway

Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox‐LDL receptor‐1 (LOX‐1) plays a central role in ox‐LDL‐mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox‐LDL through regulating eNOS activity and LOX‐1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox‐LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox‐LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre‐treatment. Meanwhile, phosphorylation of AMP‐activated protein kinase (AMPK) was suppressed by ox‐LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone‐mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone‐mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX‐1 protein expression in RMVECs was suppressed by ciglitazone, but re‐enhanced by blocking PPARγ or AMPK. Ox‐LDL‐induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX‐1. Collectively, these data demonstrate that ciglitazone‐mediated PPARγ activation suppresses LOX‐1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.     

Osteosarcoma cells induce endothelial cell proliferation during neo‐angiogenesis

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin‐dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein‐dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary‐like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1‐dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase‐3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy. J. Cell. Physiol. 228: 846–852, 2013. © 2012 Wiley Periodicals, Inc.     

Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells. Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKalpha1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK --> Rac1 --> Akt axis in the control of eNOS in endothelial cells.