Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.     

Ascorbic acid blocks the growth inhibitory effect of tumor necrosis factor-alpha on endothelial cells

Impaired endothelial cell proliferation has been proposed to be an early, critical defect contributing to the development of atherosclerosis. Recent studies show that high plasma tumor necrosis factor (TNF)-alpha levels and low serum ascorbic acid (AA) levels correlate with atherosclerosis severity. Additionally, AA has been reported to have potential beneficial effects in preventing atherosclerosis. Based on these studies, we investigated the role of AA (< or =1mM) on TNF-alpha-mediated vascular endothelial cell growth inhibition in vitro. In accordance with previous reports, we found that TNF-alpha alone inhibited endothelial cell proliferation. Further studies revealed that AA alone enhanced endothelial cell proliferation and that AA blocked endothelial cell growth inhibition induced by TNF-alpha. By contrast, we observed no effect of AA on endothelial cell activation or nuclear entry of nuclear factor-kappaB in response to TNF-alpha. The protective effect of AA on endothelial cell proliferation was not simply the result of its antioxidant activity but did correlate with collagen IV expression by endothelial cells. AA pre-treatment of proliferating endothelial cells promoted retinoblastoma protein (Rb) phosphorylation and decreased p53 levels when compared to untreated cells. Furthermore, the addition of AA to TNF-alpha-treated proliferating endothelial cells blocked both the inhibition of retinoblastoma protein phosphorylation and enhanced p53 expression induced by TNF-alpha. Consistent with these results, we found that AA protects endothelial cells against TNF-alpha-induced apoptosis. These studies highlight the potential therapeutic role of AA in promoting endothelial cell proliferation during inflammatory conditions, such as atherosclerosis and cardiovascular disease.     

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.     

Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.     

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.     

An endothelial cell genetic screen identifies the GTPase Rem2 as a suppressor of p19ARF expression that promotes endothelial cell proliferation and angiogenesis

Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53 endothelial cell arrest identified Rem2, a Ras-like GTPase. We show that ectopic Rem2 suppresses p14(ARF) (human) or p19(ARF) (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of ectopic Rem2 by RNA interference restores p19(ARF) expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19(ARF) expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is up-regulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis.     

Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.     

Importance of inverse correlation between ALDH3A1 and PPARγ in tumor cells and tissue regeneration

Aldehyde dehydrogenase (ALDH) enzymes are involved in maintaining cellular homeostasis by metabolizing both endogenous and exogenous reactive aldehydes. They modulate several cell functions including proliferation, differentiation, survival as well as cellular response to oxidative stress. We previously reported that ALDH3A1 expression is inversely correlated with the activation of PPARs (Peroxisome Proliferators-Activated Receptors), a category of orphan nuclear hormone receptors, in both rat and human cells. PPARγ is involved in cell proliferation. In this study, we have used PPARγ transfection and inhibition to examine the relationship between ALDH3A1 and PPARγ and their role as regulators of cell proliferation. Induction of PPARγ in A549 and NCTC 2544 cells by transfection caused a decrease in ALDH3A1 and inhibition of cell proliferation, a result we obtained previously using ligands that induce PPARγ. A reduction of PPARγ expression using siRNA increased ALDH3A1 expression and cell proliferation. In cells induced to proliferate in a model of tissue regeneration, ALDH3A1 expression increased during the period of proliferation, whereas PPARγ expression decreased. In conclusion, through modulation of PPARγ or ALDH3A1, it may be possible to reduce cell proliferation in tumor cells or stimulate cell proliferation in normal cells during tissue regeneration.     

Regulation of NF-kappaB activation and nuclear translocation by exogenous nitric oxide (NO) donors in TNF-alpha activated vascular endothelial cells.

Nitric oxide (NO) is a unique mediator which may promote or suppress inflammation. In this study, we examine the effect of exogenous NO on nuclear translocation of nuclear factor-kappa B (NF-kappaB) in quiescent human umbilical vein endothelial cells (HUVECs) subsequently activated by tumor necrosis factor-alpha (TNF-alpha), and in HUVECs previously activated by TNF-alpha, a model of vascular inflammation. Quiescent and activated HUVECs are exposed to exogenous NO donors of varying half-lives and the degree of NF-kappaB translocation into the nucleus determined by unique application of immunofluorescence image analysis in whole cells and correlative biochemical analysis of activated NF-kappaB proteins in the nucleus. NO donors with shorter half-lives are more effective in blocking the activation and translocation of NF-kappaB, when added to quiescent HUVECs prior to cellular activation by TNF-alpha. However, in previously activated HUVECs where NF-kappaB had relocated into the cytoplasm, addition of short half-life NO donors, but not TNF-alpha, induced re-translocation of NF-kappaB back into the nucleus sustaining the inflamed cell phenotype. These data suggest that NO as an inhibitor or activator of NF-kappaB may depend on the state of activation of vascular endothelial cells in which it contacts. Additionally, in activated cells, NO may modulate expression of NF-kappaB-dependent gene products, when cytokines are ineffective.     

High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor-alpha in human endothelial cells.

Endothelial expression of matrix metalloproteinases has been implicated in angiogenesis and endothelial cell proliferation. Recently, it has been shown that high-density lipoproteins (HDLs) promote angiogenesis. In the present study, we investigated the effects of native HDLs on the expression of several proteases and their inhibitors in human umbilical vein endothelial cells. We show that ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif) was potently induced by incubation with lipopolysaccharide or tumor necrosis factor-alpha and that the expression was significantly reduced in the presence of HDL subfraction 3. Since ADAMTS-1 has recently been shown to inhibit endothelial cell proliferation, the result of the present work may represent a new mechanism by which HDL could have a positive effect on endothelial cell and vascular wall function.     

Nuclear prostaglandin signaling system: biogenesis and actions via heptahelical receptors

Prostaglandins are ubiquitous lipid mediators that play pivotal roles in cardiovascular homeostasis, reproduction, and inflammation, as well as in many important cellular processes including gene expression and cell proliferation. The mechanism of action of these lipid messengers is thought to be primarily dependent on their interaction with specific cell surface receptors that belong to the heptahelical transmembrane spanning G protein-coupled receptor superfamily. Accumulating evidence suggests that these receptors may co-localize at the cell nucleus where they can modulate gene expression through a series of biochemical events. In this context, we have recently demonstrated that prostaglandin E2-EP3 receptors display an atypical nuclear compartmentalization in cerebral microvascular endothelial cells. Stimulation of these nuclear EP3 receptors leads to an increase of eNOS RNA in a cell-free isolated nuclear system. This review will emphasize these findings and describe how nuclear prostaglandin receptors, notably EP3 receptors, may affect gene expression, specifically of eNOS, by identifying putative transducing elements located within this organelle. The potential sources of lipid ligand activators for these intracellular sites will also be addressed. The expressional control of G-protein-coupled receptors located at the perinuclear envelope constitutes a novel and distinctive mode of gene regulation.     

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes.

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.     

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.     

Kaposi's sarcoma-associated herpesvirus activation of vascular endothelial growth factor receptor 3 alters endothelial function and enhances infection

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8) is the etiologic agent of Kaposi's sarcoma, an endothelial neoplasm. This gamma-herpesvirus encodes for several unique proteins that alter target cell function, including the virion envelope-associated glycoprotein B (gB). Glycoprotein B has an RGD (Arg-Gly-Asp) motif at the extracellular amino terminus region and binds to the alpha3beta1 surface integrin, which enhances virus entry. We now report that gB can activate the vascular endothelial growth factor receptor 3 (VEGFR-3) on the surface of microvascular endothelial cells and trigger receptor signaling, which can modulate endothelial migration and proliferation. Furthermore, we observed that VEGFR-3 expression and activation enhance KSHV infection and participate in KSHV-mediated transformation. These functional changes in the endothelium may contribute to the pathogenesis of Kaposi's sarcoma and suggest that interventions that inhibit gB activation of VEGFR-3 could be useful in the treatment of this neoplasm.     

Mapping of Human DNA-binding Nuclear Protein (NP220) to Chromosome Band 2p13.1-p13.2 and Its Relation to Matrin 3

Human NP220 (hNP220) is a novel DNA-binding nuclear protein, which has an arginine/serine-rich motif and polypyrimidine tract-binding motif, and NP220s and matrin 3 are thought to form a novel family of nuclear proteins. We have determined a chromosomal localization of the cDNA encoding human NP220 to 2p13.1-p13.2 by using fluorescence in situ hybridization. Human matrin 3 cDNA was mapped to chromosomes 1p13.1-p21.1 and 5q31.3, demonstrating that these novel nuclear proteins with similar functions are on different chromosomes.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Impact of Conditional miRNA126 Overexpression on Apoptosis-Resistant Endothelial Cell Production

The activation of endothelial cells is essential to repair damage caused by atherosclerosis via endothelial cell proliferation and migration. Overexpression of VEGF (vascular endothelial growth factor) and the downstream gene, B-cell lymphoma-2 (BCL-2) could result in apoptosis-resistant endothelial cells, which are responsible for aggravated hyperplasia and instable plaques generation. Previous studies have shown that miRNA126 could regulate the expression of VEGF. Here, we verified the existence of a miRNA126 binding site in VEGF’s 3’UTR. Additionally, VEGF regulated BCL-2 expression via AP1 (Activator Protein 1) binding site in BCL-2’s promoter. Next, we established an apoptosis-resistant endothelial cell line and constructed a lentiviral vector to express miRNA126 under the control of the BCL-2 promoter to investigate whether conditional expression of miRNA126 could modulate VEGF and BCL-2 expression in apoptosis-resistant endothelial cells. This lentiviral system specifically expressed miRNA126 in cells with high BCL-2 levels, downregulated VEGF expression, inhibited MAPK pathway activation and downregulated BCL-2 expression via suppression of AP1, and as a whole, reduced apoptosis-resistant endothelial cells, while the effects of miRNA126 on normal endothelial cells were relatively small. Our results demonstrate that conditional miRNA126 overexpression under the control of the downstream BCL-2 promoter provides a flexible regulatory strategy for reducing the apoptosis-resistant endothelial cells without having a significant impact on normal endothelial cells.