Inhibition Of Endothelial Cell Proliferation Research Articles

An Energy-Sensor Network Takes Center Stage During Endothelial Aging

See related article, pages 1384–1393 Blood vessels form a critical interface between the environment and the organism by supplying nutrients, oxygen and macromolecules to all tissues and cells in the body. Endothelial cells (ECs) line the inner surface of the hierarchically branched blood vessel network and integrate functionally into different organs to support tissue growth and function in development, physiology, and disease. In healthy postnatal tissues, ECs are mostly quiescent and rarely migrate or proliferate. However, ECs possess a remarkable phenotypic plasticity in terms of being able to proliferate rapidly in response to vascular injury, tissue ischemia, or other stress conditions. This phenotypic plasticity progressively declines when ECs age and/or are permanently exposed to metabolic (eg, hyperglycemia) and environmental stressors (eg, oxidative stress) and coincides with the development of endothelial senescence.1 Cellular senescence is a stress response that is characterized by a robust inhibition of cell proliferation, which often becomes irreversible and independent of the initial stress signal.2 Senescence can be observed in most mammalian cells after extended propagation in culture and is related to the attrition of telomeres after repetitive cycles of cell divisions.2 In addition to telomere shortening, other stressors such as DNA damage, genomic instability, and oxidative stress can induce a similar growth arrest and trigger senescence.2 Senescent cells display characteristic alterations in cell morphology and gene expression that may weaken cellular functions. In ECs, these changes cause a phenotype that is proinflammatory, prothrombotic, and proatherosclerotic, which also negatively affects the vasodilatory, angiogenic, and regenerative properties of ECs and, thus, accelerates the development of several cardiovascular diseases.1 Despite advances in understanding the molecular mechanisms of endothelial senescence and vascular aging, the signaling networks governing the development and progression of EC senescence remain less well defined. In this issue of Circulation Research …

Temporal and Spatial Regulation of Histone Deacetylase-7 and β-Catenin in Endothelial Cells

See related article, pages 1202–1211 The importance of the endothelium to cardiovascular health cannot be overstated. This cell layer is the front line for regulation of vascular tone, permeability, platelet stability, and angiogenesis, with its dysfunction touted as a harbinger of cardiovascular disease.1–3 Turnover of the endothelium in adults is relatively low, but proliferation can be triggered by inflammation, matrix disruption, or ischemia.4 Our appreciation of this cell lining is acutely apparent following percutaneous coronary intervention, where delayed reendothelialization can cause thrombosis and exacerbate restenosis.5 An even greater challenge is the aging patient with eroding endothelial function.6 Indeed, an essential goal of cardiovascular science is to better elucidate the mechanisms that govern endothelial health and renewal. In this issue of Circulation Research , Margariti et al provide evidence for a relationship between β-catenin and histone deacetylase (HDAC)7 in human endothelial cells.7 Firstly, they demonstrate that overexpression of HDAC7 results in decreased β-catenin nuclear translocation, downregulation of β-catenin target genes, decreased β-catenin activity, and an associated inhibition of endothelial cell proliferation. Conversely, HDAC7 knockdown caused β-catenin nuclear translocation, but surprisingly also caused downregulation of cell cycle genes and a decrease in proliferation. These findings led Margariti et al to discover that HDAC7 can bind directly to β-catenin in endothelial cells. Moreover, vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation was shown to be associated with HDAC7 degradation, disruption of HDAC7–β-catenin binding and β-catenin nuclear translocation (Figure A). This novel protein–protein interaction balances nuclear trafficking and cytoplasmic sequestration of β-catenin, as well as transcriptional activity of β-catenin and degradation of HDAC7 (Figure, B). This intracellular trafficking impacts endothelial proliferation and sets the stage for novel feedback mechanisms in VEGF receptor signaling and VEGF transcription. Figure. HDAC7-β-catenin interactions in VEGF-stimulated endothelial cells. A , Summary of …

Abstract 3430: Breast tumor cells inhibit vascular gap junction communication by downregulation of mural cell Connexin 43

Abstract A functional blood vessel is composed of the endothelium, the cell layer that forms the channel through which blood flows, and mural cells, the vascular smooth muscle cells (vSMC) and pericytes that serve to support and stabilize the endothelium. Proper physical and functional association between mural cells and endothelial cells (EC) causes EC to become quiescent. In contrast, the vasculature formed by a tumor is highly disorganized and exhibits decreased and abnormal association with mural cells, which allows the vessel to be unstable and proliferative. However, the mechanism(s) by which tumors prevent proper association between mural cells and the endothelium have not been well elucidated. Gap junctions (GJ) between EC and mural cells are required for EC-induced mural cell differentiation during vessel development and are involved in maintaining vasomotor tone and electrical conductance. Since GJ play an important role in cell-cell contacts and contact between mural cells and EC is disrupted in tumors, we investigated whether inhibitory mural cell interactions with EC require functional gap junction communication. Using a parachute dye transfer assay, we have demonstrated that exposure to media conditioned by MDA-MB-231 breast cancer cells substantially decreases gap junction communication between vSMC and EC. Although levels of the GJ proteins connexin 40, 43, and 45 are not altered in tumor-exposed EC, we observed that connexin 43 (Cx43) protein is downregulated in vSMC by tumor conditioned media in a time- and dose-dependent manner. A similar decrease in Cx43 is observed in response to 4T1 cells, but not MCF7 or SKBR3 cells. Quantitative real-time PCR suggests that Cx43 loss results at least partially from a decrease in Cx43 mRNA levels. Pharmacological inhibitors of MAPK, Src, and PKC indicate that these signaling pathways are not involved in tumor-induced Cx43 downregulation. We further demonstrate that expression of Cx43 by mural cells is required for the mural cell-induced inhibition of EC proliferation, as mural cells in which Cx43 levels have been reduced by Cx43 siRNA transfection have decreased ability to attenuate EC proliferation compared to control siRNA-transfected mural cells. This effect is observed with both undifferentiated mural precursor C3H10T1/2 cells as well as fully differentiated vSMC mural cells. These data suggest that tumor-induced loss of mural cell Cx43 may both disrupt mural cell-EC contacts in a differentiated, mature vessel to allow for initiation of angiogenesis, as well as prevent proper association of mural cell precursors with a newly formed tumor vessel to prevent termination of angiogenesis. Restoration of Cx43-based junctional contacts between mural cells and EC to induce EC quiescence may therefore represent a novel strategy to elicit vascular normalization and inhibition of tumor angiogenesis. (Supported by NIH CA138727) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3430.

Expression and purification of soluble recombinant Human Endostatin in Escherichia coli

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.

Activation of the planar cell polarity formin DAAM1 leads to inhibition of endothelial cell proliferation, migration, and angiogenesis

The Wnt/planar cell polarity (PCP) pathway regulates directed cell movement during development and was recently found to play a critical role in endothelial cell proliferation and angiogenesis [Zhang Y, et al. (2006) Chem Biol 13:1001-1009; Masckauchan TN, et al. (2006) Mol Biol Cell 17:5163-5172]. However, the mechanisms by which PCP signaling components regulate angiogenesis remain unknown. We report that expression of a constitutively active C-terminal domain of Dishevelled-associated activator of morphogenesis 1 (DAAM1) selectively inhibited endothelial cell proliferation. Moreover, this activated construct suppressed endothelial cell migration and the ability to form coordinated networks in vivo and in vitro. Although constitutively active DAAM1 (CDAAM1) induced both actin polymerization and microtubule (MT) stabilization, the stabilization of MTs alone was sufficient to inhibit endothelial cell growth selectively. Inhibition of actin polymerization alone by jasplakinolide treatment failed to reproduce the inhibitory effects of CDAAM1. These results indicate that DAAM1 regulates endothelial cell growth through MT stabilization in a cell type-selective manner and suggest that PCP signaling plays a pivotal role in angiogenesis by regulating MT stabilization.

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm(2), 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3'-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Sprouty1 inhibits angiogenesis in association with up-regulation of p21 and p27

Sprouty1 (Spry1) is a conserved antagonist of FGF signaling. The goal of this study was to further explore the downstream mechanisms governing Spry1 inhibition of endothelial cell proliferation. Up-regulation of Spry1 in HUVECs inhibited tube formation on Matrigel (n = 6, P < 0.001). This was associated with decreased proliferation as measured by BrdU incorporation (n = 6, P < 0.001) and increased protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A), p21 and cyclin-dependent kinase inhibitor 1B (CDKN1B), p27. A transcriptional analysis using a targeted human angiogenesis array following up-regulation of Spry1 demonstrated a >2-fold increase in an anti-angiogenic factor, serpin peptidase inhibitor, clad F (Serpinf1), and a >2-fold decrease in pro-angiogenic factors fms-related tyrosine kinase 1 (FLT1), angiopoietin2 (Ang-2), and placental growth factor (PGF) (n = 2). To define upstream mechanisms that may regulate endogenous Spry1, we performed a search for responsive elements upstream of the promoter region. This search resulted in the identification of multiple degenerate hypoxia responsive elements. Exposure to hypoxia resulted in a significant increase in Spry1 expression (n = 8, P < 0.01). These findings shed new light on downstream signaling pathways associated with Spry1 anti-proliferative responses, and provide new evidence that hypoxia stimulates Spry1 expression.

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

Nanoparticle-mediated targeting of phosphatidylinositol-3-kinase signaling inhibits angiogenesis

Dysregulation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a hallmark of human cancer, occurring in a majority of tumors. Activation of this pathway is critical for transformation and also for the angiogenic switch, which is a key step for tumor progression. The objective of this study was to engineer a PI3K inhibitor-loaded biodegradable nanoparticle and to evaluate its efficacy. Here we report that a nanoparticle-enabled targeting of the PI3K pathway results in inhibition of downstream Akt phosphorylation, leading to inhibition of proliferation and induction of apoptosis of B16/F10 melanoma. It, however, failed to exert a similar activity on MDA-MB-231 breast cancer cells, resulting from reduced internalization and processing of nanoparticles in this cell line. Excitingly, the nanoparticle-enabled targeting of the PI3K pathway resulted in inhibition of endothelial cell proliferation and tubulogenesis, two key steps in tumor angiogenesis. Furthermore, it inhibited both B16/F10- and MDA-MB-231-induced angiogenesis in a zebrafish tumor xenotransplant model. Our study, for the first time, shows that targeting of the PI3K pathway using nanoparticles can offer an attractive strategy for inhibiting tumor angiogenesis.

Anti-vascular endothelial growth factor (VEGF) specific activity of intravenous immunoglobulin (IVIg)

Intravenous immunoglobulins (IVIg) preparations can be beneficial therapeutic agents for the treatment of tumor metastases as has been shown in both human and animal studies. Operating mechanisms have not yet been completely elucidated. Some of the mechanisms proposed entail the stimulation of the production of IL-12, a cytokine that exhibits anti-angiogenic activities, as well as inhibition of endothelial cells proliferation and vascular endothelial growth factor (VEGF) secretion. The aim of the present study was to investigate whether in an IVIg preparation there are natural antibodies directed against VEGF with the potential to affect angiogenesis. Using both sandwich and direct ELISA assays, IVIg was found to specifically recognize and bind VEGF in a dose-dependent manner. The binding specificity was confirmed by inhibition of IVIg binding to VEGF by VEGF as an inhibitor, as shown by ELISA and immunoblot. A mouse hind limb ischemia model was employed to evaluate the in vivo IVIg-induced inhibition of angiogenesis. IVIg was found to exhibit inhibitory effect on VEGF-mediated blood perfusion in the ischemic limb. The present study shows a presence of anti-VEGF fraction in IVIg preparation.

Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection

Vascular endothelium is vulnerable to the attack of glucose-derived oxoaldehydes (glyoxal and methylglyoxal) during diabetes, through the formation of advanced glycation end products (AGEs). Although aminoguanidine (AG) has been shown to protect against the AGE-induced adverse effects, its protection against the glyoxal-induced alterations in vascular endothelial cells (ECs) such as cytotoxicity, barrier dysfunction, and inhibition of angiogenesis has not been reported and we investigated this in the bovine pulmonary artery ECs (BPAECs). The results showed that glyoxal (1-10 mM) significantly induced cytotoxicity and mitochondrial dysfunction in a dose- and time-dependent (4-12 h) fashion in ECs. Glyoxal was also observed to significantly inhibit EC proliferation. The study also revealed that glyoxal induced EC barrier dysfunction (loss of trans-endothelial electrical resistance), actin cytoskeletal rearrangement, and tight junction alterations in BPAECs. Furthermore, the results revealed that glyoxal significantly inhibited in vitro angiogenesis on the Matrigel. For the first time, this study demonstrated that AG significantly protected against the glyoxal-induced cytotoxicity, barrier dysfunction, cytoskeletal rearrangement, and inhibition of angiogenesis in BPAECs. Therefore, AG appears as a promising protective agent in the treatment of AGE-induced vascular endothelial alterations and dysfunction during diabetes, presumably by blocking the reactivity of the sugar-derived dicarbonyls such as glyoxal and preventing the formation of AGEs.

Cadmium Is a Novel and Independent Risk Factor for Early Atherosclerosis Mechanisms and In Vivo Relevance

Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.-2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm(2), P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

CCR3 is a target for age-related macular degeneration diagnosis and therapy

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularization (CNV). We report that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in CNV endothelial cells in humans with AMD, and that, despite the expression of its ligands eotaxin-1, -2, and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation as it occurred in mice lacking eosinophils or mast cells and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor-A (VEGF-A) neutralization, which is currently in clinical use, and, unlike VEGF-A blockade, not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Necessary for Endothelial Cell Proliferation

Recent breakthroughs in the store-operated calcium (SOC) entry pathway have identified Stim1 as the endoplasmic reticulum (ER) calcium sensor and Orai1 as the pore forming subunit of the highly calcium selective CRAC channel. Previous studies have suggested that endothelial cell (EC) SOC is encoded by members of the Canonical Transient Receptor Potential (TRPC) channel family, either TRPC1 or TRPC4. Here we show that passive store depletion or receptor activation by thrombin or VEGF activates SOC entry pathway in primary EC with classical SOC pharmacological features. EC possess the archetypical store-depletion activated CRAC current. By amplifying currents in divalent free bath solutions, we show that EC CRAC has similar characteristics to that recorded from RBL cells, namely a similar time course of activation, sensitivity to 2-APB and low concentrations of lanthanides, the same inwardly rectifying I/V relationship, very positive reversal potential, and large sodium currents displaying the typical phenomenon of depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOC entry and CRAC currents in EC which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, complete knockdown of either TRPC1 or TRPC4 proteins had no effect on SOC entry in EC. Smaller CRAC current densities in EC compared to those recorded in RBL cells were due to lower expression of Stim1. Ectopic expression of Stim1 in EC increased their CRAC currents to a size comparable to those in RBL cells. Knockdown of either Stim1, Stim2 or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim1. These results are first to establish the requirement of Stim1/Orai1 in the endothelial SOC pathway necessary for proliferation.

A spatial model of tumor-host interaction: application of chemotherapy.

In this paper we consider chemotherapy in a spatial model of tumor growth. The model, which is of reaction-diffusion type, takes into account the complex interactions between the tumor and surrounding stromal cells by including densities of endothelial cells and the extra-cellular matrix. When no treatment is applied the model reproduces the typical dynamics of early tumor growth. The initially avascular tumor reaches a diffusion limited size of the order of millimeters and initiates angiogenesis through the release of vascular endothelial growth factor (VEGF) secreted by hypoxic cells in the core of the tumor. This stimulates endothelial cells to migrate towards the tumor and establishes a nutrient supply sufficient for sustained invasion. To this model we apply cytostatic treatment in the form of a VEGF-inhibitor, which reduces the proliferation and chemotaxis of endothelial cells. This treatment has the capability to reduce tumor mass, but more importantly, we were able to determine that inhibition of endothelial cell proliferation is the more important of the two cellular functions targeted by the drug. Further, we considered the application of a cytotoxic drug that targets proliferating tumor cells. The drug was treated as a diffusible substance entering the tissue from the blood vessels. Our results show that depending on the characteristics of the drug it can either reduce the tumor mass significantly or in fact accelerate the growth rate of the tumor. This result seems to be due to complicated interplay between the stromal and tumor cell types and highlights the importance of considering chemotherapy in a spatial context.

Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities

The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails.

Enantioselective Synthesis of (+)-Cortistatin A, a Potent and Selective Inhibitor of Endothelial Cell Proliferation

This manuscript describes an enantioselective synthesis of the naturally occurring inhibitor of endothelial cell proliferation, cortistatin A. Key steps of the synthesis are a silicate-directed elimination/ring expansion reaction and a highly diastereoselective aza-Prins cyclization with a subsequent transannular etherification.

Dual Inhibition of PI3Kα and mTOR as an Alternative Treatment for Kaposi's Sarcoma

Rapamycin (or sirolimus), the prototypical inhibitor of the mammalian target of rapamycin (mTOR) and an immunosuppressant used for the prevention of renal transplant rejection, has recently emerged as an effective treatment for Kaposi's sarcoma (KS), an enigmatic vascular tumor and a model for pathologic angiogenesis. Indeed, recent work supports a role for mTOR as a central player in the transformation of endothelial cells by the KS-associated herpesvirus-encoded G protein-coupled receptor (vGPCR), the viral oncogene believed to be responsible for causing KS. However, emerging evidence that rapamycin may transiently promote the activation of Akt may limit its use as an anti-KS therapy. Here, we show that activation of Akt in endothelial cells expressing vGPCR is augmented by treatment with rapamycin, resulting in the up-regulation of several Akt proliferative and survival pathways. However, use of a novel dual phosphatidylinositol 3-kinase alpha (PI3Kalpha)/mTOR inhibitor, PI-103, effectively and independently blocked activation of both PI3K and mTOR in vGPCR-expressing endothelial cells. This resulted in more effective inhibition of endothelial cell proliferation and survival in vitro and tumor growth in vivo. Our results suggest that PI-103 may be an effective therapeutic option for the treatment of patients with KS. Moreover, as KS may serve as a model for pathologic angiogenesis, our results further provide the basis for the early assessment of PI-103 as an antiangiogenic chemotherapeutic.

Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

A systematic methodology for proteome-wide identification of peptides inhibiting the proliferation and migration of endothelial cells

We introduce a systematic computational methodology based on bioinformatics that has enabled us to identify and classify >120 endogenous peptide inhibitors of endothelial cell proliferation and migration. These peptides are derived from members of the type IV collagen, thrombospondin, and CXC chemokine protein families, as well as somatotropin hormones, serpins, and various kringle-containing proteins. Their activity in suppressing the proliferation and migration of endothelial cells in vitro provides proof of principle for the validity of this computational method. Interestingly, some of the peptides are derived from proteins known to be proangiogenic. By performing receptor neutralization studies, we have identified receptors to which these peptides bind. On the basis of this receptor-binding information, we evaluated several examples of peptide-based combinatorial screening strategies. In some cases, this combinatorial screening identified strong synergism between peptides. The current work provides a guideline for a computational-based peptidomics approach for the discovery of endogenous bioactive peptides.