Endothelial Cell Growth Arrest Research Articles

NG2 as an Identity and Quality Marker of Mesenchymal Stem Cell Extracellular Vesicles.

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2+ cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

Pericyte chemomechanics and the angiogenic switch: insights into the pathogenesis of proliferative diabetic retinopathy?

To establish the regulatory roles that pericytes have in coordinating retinal endothelial cell (EC) growth and angiogenic potential. Pericytes were derived from donor diabetic (DHuRP) or normal (NHuRP) human retinae, and characterized using vascular markers, coculture, contraction, morphogenesis, and proliferation assays. To investigate capillary "cross-talk," pericyte-endothelial coculture growth, and connexin-43 (Cx43) expression assays were performed. Paracrine effects were examined via treating EC with pericyte-derived conditioned media (CM) in proliferation, angiogenesis, and angiocrine assays. The effects of sphingosine 1-phosphate (S1P) were assessed using receptor antagonists. The DHuRP exhibit unique proliferative and morphologic properties, reflecting distinctive cytoskeletal and isoactin expression patterns. Unlike NHuRP, DHuRP are unable to sustain EC growth arrest in coculture and display reduced Cx43 expression. Further, CM from DHuRP (DPCM) markedly stimulates EC proliferation and tube formation. Treatment with S1P receptor antagonists mitigates DPCM growth-promotion in EC and S1P-mediated pericyte contraction. Angiocrine assays on normal and diabetic pericyte secretomes reveal factors involved in angiogenic control, inflammation, and metabolism. Effects from the diabetic microenvironment appear sustainable in cell culture: pericytes derived from diabetic donor eyes seemingly possess a "metabolic memory" in vitro, which may be linked to original donor health status. Diabetes- and pericyte-dependent effects on EC growth and angiogenesis may reflect alterations in bioactive lipid, angiocrine, and chemomechanical signaling. Altogether, our results suggest that diabetes alters pericyte contractile phenotype and cytoskeletal signaling, which ultimately may serve as a key, initiating event required for retinal endothelial reproliferation, angiogenic activation, and the pathological neovascularization accompanying proliferative diabetic retinopathy.

Stachydrine ameliorates high-glucose induced endothelial cell senescence and SIRT1 downregulation

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases.

SIRT1 Regulates Hyperglycemia-Induced Vascular Senescence

Background: Vascular senescence reflects the limited ability of vascular cells to divide and proliferate and is accompanied by specific phenotypic changes in morphology, gene expression and function. In endothelial cells, these changes result in a phenotype that is pro-inflammatory, pro-atherosclerotic, and prothrombotic. Hyperglycemia-mediated oxidative stress elicits irreversible growth arrest in endothelial cells, which is referred as “stress-induced premature senescence”. Endothelial cell senescence has recently been postulated as an important cause of type-2 diabetes-associated vascular aging. High glucose-induced endothelial senescence exaggerates vascular inflammation and thrombosis in the vessels, promoting the development of diabetic-associated cardiovascular events. Sirtuin 1 (SIRT1) is highly expressed in vasculature and distinctively controls angiogenic signaling in endothelial cells. Recently, SIRT1 has been identified as an important regulator of endothelial cell senescence-like growth arrest and dysfunction. However, the mechanism underlying the high glucose induced endothelial cell senescence leading to vascular dysfunction is not fully understood. Methods: Mouse microvessel endothelial cells were exposed to normal (11mM) and high glucose (40mM) for 72 hrs. The level of SIRT1, p21 and phospho-acetylated status of Foxo1 were measured by immunoblotting. To measure senescence, cells were fixed and stained for senescence-associated B-galactosidase activity. The percentage of B-galactosidase positively stained (blue color) cells was counted. Results: Immunoblot analysis reveals that high glucose exposure caused a significant reduction of SIRT1 level along with altered phospho-acetylation levels of Foxo1 proteins. Furthermore, high glucose treatment increased the percentage of senescence-associated B-galactosidase activity in endothelial cells. Conclusion: Collectively, these data suggest that high glucose induced depletion of SIRT1 plays a crucial role in diabetes-associated endothelial cell senescence and strengthens concept that activation of SIRT1 is an important target for pharmaco-therapy for diabetes-associated endothelial dysfunction and vascular aging. This abstract is part of project funded by: NPRP: 08-165-3-054

Reversal of TNP-470-Induced Endothelial Cell Growth Arrest by Guanine and Guanine Nucleosides

The mechanism of action of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21(WAF1/CIP1), respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21(WAF1/CIP1) by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid, showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced after TNP-470 treatment. Addition of guanosine at the start of G(1) phase causes a doubling in GTP levels that persists to the G(1)/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather than a restoration of a drug-induced GTP deficit. Determining the mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.

A role for planar cell polarity signaling in angiogenesis

The planar cell polarity (PCP) pathway is a highly conserved signaling cascade that coordinates both epithelial and axonal morphogenic movements during development. Angiogenesis also involves the growth and migration of polarized cells, although the mechanisms underlying their intercellular communication are poorly understood. Here, using cell culture assays, we demonstrate that inhibition of PCP signaling disrupts endothelial cell growth, polarity, and migration, all of which can be rescued through downstream activation of this pathway by expression of either Daam-1, Diversin or Inversin. Silencing of either Dvl2 or Prickle suppressed endothelial cell proliferation. Moreover, loss of p53 rescues endothelial cell growth arrest but not the migration inhibition caused by PCP disruption. In addition, we show that the zebrafish Wnt5 mutant (pipetail (ppt)), which has impaired PCP signaling, displays vascular developmental defects. These findings reveal a potential role for PCP signaling in the coordinated assembly of endothelial cells into vascular structures and have important implications for vascular remodeling in development and disease.

A Novel Application for Murine Double Minute 2 Antagonists

Impairment of the p53 tumor suppressor network1 is thought to be involved in a large percentage of tumors either by mutations in the p53 gene2–4 or by increased expression of its major control system, the MDM25 (murine double minute 2; HDM2 for its human equivalent). It is important to realize that levels of p53 are subjected to an autoregulatory feedback loop by MDM26,7 as p53 upregulates MDM2 gene expression and MDM2 protein in turn binds to p53. MDM2 is an E3 ubiquitin ligase and transports p53 to the cytoplasm where it promotes p53 ubiquitination and degradation by the proteasome. In human cancer, increased levels of MDM26 are caused either by gene amplification, increased expression induced by activated p53, stabilization by an aberrantly spliced form of HMDX8, or augmented translation. In addition to these mechanisms, functional single-nucleotide polymorphisms (SNP) such as the 1 at nucleotide 309 (SNP309) in the MDM2 gene can modulate MDM2 expression and cause increased tumor progression.9 …

Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene (MAP2) resulted in an early gastrulation defect, which is bypassed in double MetAP-2/p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.

Functionally novel tumor necrosis factor-alpha-modulated CHR-binding protein mediates cyclin A transcriptional repression in vascular endothelial cells.

Local expression of tumor necrosis factor-alpha (TNF-alpha) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-alpha induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-alpha-mediated suppression of cyclin A in ECs. TNF-alpha exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-alpha inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (Cell cycle-Dependent Elements-Cell cycle genes Homology Region) region of cyclin A promoter as a target for TNF-alpha suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-alpha-mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-alpha was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-alpha was protein synthesis dependent. Additionally, exposure of ECs to TNF-alpha markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.

Modulation of endothelial cell growth arrest and apoptosis by vascular endothelial growth inhibitor.

Vascular endothelial growth inhibitor (VEGI), a new member of the tumor necrosis factor family, is an endothelial cell-specific gene and a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. We report here that VEGI mediates the following two activities in endothelial cells: early G(1) arrest in G(0)/G(1) cells responding to growth stimuli, and programmed death in proliferating cells. G(0)/G(1)-synchronized bovine aortic endothelial cells were treated with VEGI before and after the onset of the growth cycle. When the cells were stimulated with growth conditions but treated simultaneously with VEGI, a reversible, early-G(1) growth arrest occurred, evidenced by the lack of late G(1) markers such as hyperphosphorylation of the retinoblastoma gene product and upregulation of the c-myc gene. Additionally, VEGI treatment led to inhibition of the activities of cyclin-dependent kinases CDK2, CDK4, and CDK6. In contrast, VEGI treatment of cells that had entered the growth cycle resulted in apoptotic cell death, as evidenced by terminal deoxytransferase labeling of fragmented DNA, caspase 3 activation, and annexin V staining, all of which were lacking in nonproliferating cells treated with VEGI. Additionally, stress-signaling proteins p38 and JNK were not as fully activated by VEGI in quiescent as compared with proliferating populations. These findings suggest a dual role for VEGI, the maintenance of growth arrest and induction of apoptosis, in the modulation of the endothelial cell cycle.

Transcriptional Up-regulation of p27Kip1 during Contact-Induced Growth Arrest in Vascular Endothelial Cells

By plating porcine aortic endothelial cells at two different densities and thereby inducing two different time courses of contact-induced growth arrest, the temporal correlation between p27Kip1 expression and cell cycle progression was investigated. When the quiescent cells were replated, they synchronously entered S phase with a peak at 20 h in both cases, while the cells plated at 25 and 80% of confluent densities exited the cell cycle by 96 and 48 h, respectively. Nuclear p27Kip1 disappeared when the cells reentered the cell cycle and then recovered when the cells exited the cell cycle. The change in p27Kip1 was associated with a concomitant change in Kip1 mRNA. The p27Kip1 degradation activity did not increase in the cells reentering the cell cycle, nor did it decrease in the cells exiting the cell cycle. The Kip1 mRNA stability decreased in the growing cells and increased in the cells exiting the cell cycle and at confluence. A nuclear run-on assay revealed a close correlation between the Kip1 transcriptional activity and the level of Kip1 mRNA. We conclude that the cell–cell contact up-regulated the Kip1 gene transcription and increased the Kip1 mRNA stability, which was related to the recovery of p27Kip1 protein during contact-induced growth arrest in endothelial cells.

Extracellular Matrix-Derived Angiogenic Factor(s) Inhibit Endothelial Cell Proliferation, Enhance Differentiation, and Stimulate Angiogenesis in vivo

To isolate matrix molecules with angiogenic activity, tumor extracellular matrix (ECM) fractions from the basement membrane preparation Matrigel were analyzed for effects on endothelial cell (EC) proliferation, differentiation, and vessel formation in vivo. Inhibition of human and bovine EC DNA synthesis was evident upon treatment with several soluble Matrigel fractions including conditioned media (MGCM). After size fractionation of MGCM, EC growth arrest was activated by factor(s) smaller than 3,000 daltons (3KF). Bovine EC differentiation (tube formation) was promoted by both MGCM and 3KF fractions in two different models using matrigel or collagen gels to stimulate tube formation. The 3KF factor(s) stimulated angiogenesis when implanted in the cornea or subcutaneously in mice. FGF-induced angiogenesis and blood flow were increased in the presence of 3KF factor(s), an effect that was inhibited by the anti-angiogenic molecule endostatin. Further characterization of the low molecular weight 3KF samples by RP-HPLC revealed several fractions exhibiting EC growth arrest activity. These results suggest that the ability of ECM preparations to induce EC growth arrest and tube formation may reside, at least partially, in previously undetected low molecular weight molecules. Characterization of these ECM-associated inhibitors may lead to the development of novel anti-angiogenic and anti-tumor compounds.

Levamisole induced apoptosis in cultured vascular endothelial cells.

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5 - 2 mmol l(-1)) alone or in combination with antioxidants (10 mmol l(-1) glutathione or 5 mmol l(-1) N-Acetylcysteine or 0.1 mmol l(-1) Tocopherol) were evaluated for apoptosis ((3)H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation ((3)H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (-70%), reduced expression of survival factors such as clusterin (-30%), endothelin-1 (-43%), bcl-2 (-34%), endothelial NO-synthase (-32%) and pRb (Retinoblastoma protein: -89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l(-1))-induced apoptosis was inhibited by glutathione (-50%) and N-Acetylcysteine (-36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.

The antiangiogenic agent TNP-470 requires p53 and p21CIP/WAF for endothelial cell growth arrest.

Targeting the endothelial cell cycle as an antiangiogenic strategy has been difficult given the ubiquitous expression of critical cell cycle regulators. Here, we show that the antiangiogenic drug TNP-470 displays striking cell-type specificity insofar as it induces the expression of p21(CIP/WAF), a cyclin-dependent kinase inhibitor, in endothelial cells but not in embryonic or adult fibroblasts. Moreover, primary endothelial cells isolated from p53(-/-) and p21(CIP/WAF-/-) mice are resistant to the cytostatic activity of TNP-470. We also demonstrate that p21(CIP/WAF-/-) mice are resistant to the antiangiogenic activity of TNP-470 in the basic fibroblast growth factor corneal micropocket angiogenesis assay. We conclude that TNP-470 induces p53 activation through a unique mechanism in endothelial cells leading to p21(CIP/WAF) expression and subsequent growth arrest.