Role of MARCKS in regulating endothelial cell proliferation

Myristoylated alanine-rich C kinase substrate (MARCKS), as a specificprotein kinase C (PKC) substrate, mediates PKC signaling through itsphosphorylation and subsequent modification of its association withfilamentous actin (F-actin) and calmodulin (CaM). PKC has long beenimplicated in cell proliferation, and recent studies have suggestedthat MARCKS may function as a cell growth suppressor. Therefore, in thepresent study, we investigated MARCKS protein expression, distribution,and phosphorylation in preconfluent and confluent bovine pulmonarymicrovascular endothelial cells (BPMEC) in the presence or absence ofthe vascular endothelial growth factor (VEGF). In addition, we examinedfunctional alterations of MARCKS in these cells by studying theassociation of MARCKS with F-actin and CaM-dependent myosin light chain(MLC) phosphorylation. Our results indicate that MARCKS protein isdownregulated during BPMEC proliferation. Decreased MARCKSassociation with F-actin, increased actin polymerization, andCaM-dependent MLC phosphorylation appear to mediate cell shape changesand motility during BPMEC growth. In contrast, VEGF stimulated MARCKSphosphorylation without alteration of protein expression during BPMECproliferation, which may result in reduced interaction between MARCKSand actin or CaM, leading to actin reorganization and MLCphosphorylation. Our data suggest a regulatory role of MARCKS duringendothelial cell proliferation.

     

TGF-beta2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-beta2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-beta2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-beta2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-beta2 and FGF-2 oppositely affect BCE cell proliferation and TGF-beta2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-beta2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-beta2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-beta2-induced suppression of the PI3-kinase/AKT signaling pathway.     

Role of extracellular signaling on endothelial cell proliferation and protein N-glycosylation

In vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine-linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in G0/G1 phase. The cellular response to protein N-glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS-PAGE indicated that glycosylation of 80-120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry "high mannose" and "complex" type oligosaccharide chains attached to their protein core.     

17beta-estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells

It has been well documented previously that 17beta-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2 in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2 (1-100 nM), but not 17alpha-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2 was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 microM). E2 also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2 and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2 inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.     

Role of ephrin B2 in human retinal endothelial cell proliferation and migration

This study was designed to determine the presence of Eph B4 or ephrin B2 in human retinal endothelial cells (REC) and their signal transduction. Human retinal endothelial cells were stimulated with an Eph B4/Fc chimera and probed for phosphorylation of phosphatidylinositol-3-kinase (PI3K), Src, and mitogen-activated protein kinase (MAPK) pathways. Proliferation and migration were investigated after Eph B4/Fc stimulation in the presence of various pathway inhibitors. Human retinal endothelial cells express ephrin B2, with little expression of Eph B4. Treatment with EphB4/Fc chimera resulted in activation of PI3K, Src, and MAPK pathways. Eph B4-stimulated endothelial cell proliferation was mediated via PI3K, nitric oxide synthase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Blockade of Src-PI3K pathways produced significant attenuation of Eph B4/Fc-stimulated migration. These results demonstrate for the first time that ephrin B2 is present in human retinal endothelial cells. Additionally, it appears that vascular growth may be modulated in the retina through activation of the PI3K pathway and its downstream components.     

Effects of different types of fluid shear stress on endothelial cell proliferation and survival

We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.     

CaM kinase control of AKT and LNCaP cell survival

AKT and its substrate BAD have been shown to promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. The LNCaP prostate cancer cells express the carbachol-sensitive M(3) -subtype of G protein-coupled receptors that cause increases in intracellular calcium and activate the family of Ca(2+) /calmodulin-dependent protein kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), phosphorylates several substrates including AKT on threonine 308. AKT phosphorylation and activation enhances cell survival through phosphorylation of BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM KK may be mediating carbachol's activation of AKT and cell survival. Our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK and its phosphorylation of AKT. The bacterial toxin anisomycin triggered caspase-3 activation in LNCaP cells that was blocked by carbachol in a CaM KK- and AKT-dependent manner. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO-609, as well as siRNA directed against CaM KK. BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT-X, as well as siRNA to AKT. Additionally, epinephrine promoted LNCaP cell survival through activation of AKT that was insensitive to STO-609. Taken together these data suggest a survival role for CaM KK operating through AKT and BAD in LNCaP prostate cancer cells.     

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38+/-6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 microM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration.     

The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1

Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.     

Role of PLCgamma and Ca(2+) in VEGF- and FGF-induced choroidal endothelial cell proliferation

Although bothvascular endothelial growth factor (VEGF) and fibroblast growth factor(FGF) receptors have been shown to be important in the regulation ofvascular endothelial cell growth, the roles of phospholipase C (PLC)and Ca²⁺ in their downstream signaling cascades are stillnot clear. We have examined the effects of VEGF and FGF on PLCphosphorylation and on changes in intracellular Ca²⁺ levelsin primary endothelial cells. VEGF stimulation leads to PLCactivation and increases in intracellular Ca²⁺, which arecorrelated with mitogen-activated protein (MAP) kinase (MAPK)activation and cell growth. Inhibition of Ca²⁺ increases bythe Ca²⁺ chelator1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid(BAPTA)-AM resulted in marked inhibition of MAPK activation, which wasshown to be linked to regulation of cell growth in these cells. Incontrast, FGF stimulation did not lead to PLC activation or tochanges in intracellular Ca²⁺ levels, although MAPKphosphorylation and stimulation of cell proliferation were observed.Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on theseFGF-stimulated responses. These data demonstrate a direct role forPLC and Ca²⁺ in VEGF-regulated endothelial cell growth,whereas this signaling pathway is not linked to FGF-mediated effects inprimary endothelial cells. Thus endothelial cell-specific factorsregulate the ability of VEGF receptors and FGF receptors to couple tothis signaling pathway.

     

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.     

Endogenous regulation of endothelial cell proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

Role of ErbB2 mediated AKT and MAPK pathway in gall bladder cell proliferation induced by argemone oil and butter yellow

The effect of noncytotoxic doses of argemone oil (AO) and butter yellow (BY), the common adulterants in edible oil, on free radical generation and signaling pathway for cell proliferation in primary cells of gall bladder (GB) was undertaken. AO and BY showed no cytotoxicity at 0.1 μl/ml and 0.1 μg/ml concentration, respectively. AO caused significant increase in ROS after 30 min and RNS after 24 h in GB cells while no change was observed following BY treatment. Enhanced level of COX-2 was observed following AO (0.1 μl/ml) and BY (0.1 μg/ml) treatment to cells for 24 h. AO treatment caused phosphorylation of ErbB2, AKT, ERK, and JNK along with increased thymidine uptake indicating cell proliferation ability in GB cells. BY treatment also showed significant expression of these proteins with the exception of phosphorylated JNK. These results suggest that AO and BY have cell proliferative potential in GB cells following up-regulation of COX-2 and ErbB2; however, their downstream signaling molecules and free radical generation have differential response, indicating that the mechanism of proliferation is different for both compounds and may have relevance in gall bladder cancer.     

Hemin promotes proliferation and differentiation of endothelial progenitor cells via activation of AKT and ERK

Increased neovascularization is commonly observed in hemorrhagic plaques and associated with rupture of atherosclerotic lesions. This study aims to investigate whether hemin accumulated at the site of hematoma promotes neovascularization through affecting the growth and function of endothelial progenitor cells (EPCs) and the possible mechanism involved. Here we demonstrated that hemin promoted a significant increase in neovessel formation in matrigel plugs embedded in vivo and enhanced the proliferation and endothelial gene expression in EPCs in vitro. VEGF‐induced migration response and the capability to incorporate into the vascular networks were markedly enhanced in hemin‐treated EPCs. Hemin induced the phosphorylation of ERK and AKT but not p38 or JNK. The inhibition of AKT or ERK activation significantly attenuated the effect of hemin on cell proliferation. However, the enhanced migration response induced by hemin was significantly suppressed by the inhibition of AKT but not ERK. Hemin induced significant increase in reactive oxygen species (ROS) production and hemin‐induced angiogenic response of EPCs was substantially reduced by treatment with N‐acetylcysteine. Collectively, these data support that hemin‐induced ROS mediates the activation of AKT and ERK signaling pathways, which in turn promotes the cell proliferation and function of EPCs. J. Cell. Physiol. 219: 617–625, 2009. © 2009 Wiley‐Liss, Inc.     

IL-8 directly enhanced endothelial cell survival,proliferation, and matrix metalloproteinases production and regulated angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-x(L):Bcl-x(S) and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.     

Nitric oxide-an endothelial cell survival factor

Due to its unique position in the vessel wall, the endothelium acts as a barrier and thereby controls adhesion, aggregation and invasion of immune competent cells. Apoptosis of endothelial cells may critically disturb the integrity of the endothelial monolayer and contribute to the initiation of proinflammatory events. Endothelial cell apoptosis is counteracted by nitric oxide synthesised by the endothelium nitric oxide synthase (eNOS). Thus, nitric oxide inhibits endothelial cell apoptosis induced by proinflammatory cytokines and proatherosclerotic factors including reactive oxygen species and angiotensin II. The apoptosis-suppression may contribute to the profound anti-inflammatory and anti-atherosclerotic effects of endothelial-derived NO. Furthermore, the support of endothelial cell survival by NO may further play a central role for the pro-angiogenic effects of NO.     

Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)–specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.     

Role of proliferation in LAK cell development

Summary Lymphokine-activated killer (LAK) cell activity may be largely the result of activation and/or expansion of peripheral blood natural killer cells by culture with interleukin-2 (IL-2). We have examined the role of proliferation in LAK cell development by either inhibiting or enhancing the proliferative potential of peripheral blood lymphocytes. Inhibition of proliferation was accomplished using irradiation, mitomycin C, or the iron chelator deferoxamine. For each of these agents, a dose-dependent inhibition of proliferation was observed. At doses of inhibitor which nearly completely blocked thymidine uptake, the development of LAK activity was only partially impaired. The mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A) augmented the proliferative response of peripheral blood lymphocytes to IL-2. However, augmentation by PHA, but not Con A, consistently resulted in a decrease in LAK activity. This inhibition of LAK activity by PHA did not appear to be due to inhibition of the effector cell, nor to preferential expansion of irrelevant cells. These data suggests that not all LAK activity is dependent on proliferation, and that high levels of proliferation in the presence of IL-2 do not necessarily lead to LAK activity.This work was supported in part by U.S. Public Health Service Grant CA-34442 (S. H. G.) and pre-doctoral training grant CA-09120 (F. J. R.)