Apoptosis Of HUVECs Research Articles

Involvement of anion exchanger-2 in apoptosis of endothelial cells induced by high glucose through an mPTP-ROS-Caspase-3 dependent pathway

Excess apoptosis of endothelial cells (EC) plays crucial roles in the onset and progression of vasculopathy in diabetes mellitus. Anion exchanger-2 (AE2) might be involved in the vasculopathy. However, little is known about the molecular mechanisms that AE2 mediated the apoptosis of EC. The purpose of this study was to explore the role of AE2 in the apoptosis of HUVECs induced by high glucose (HG) and its possible mechanisms. First, HUVECs were exposed to different glucose concentrations (5.5, 17.8, 35.6, 71.2 and 142.4 mmol/l, respectively, pH = 7.40) for different time points (12, 24, 48, 72, 120, and 168 h, respectively). Intracellular Cl(-) concentration ([Cl(-)]i), AE2 expression and the apoptosis were assayed. Then, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), Cl(-)-free media or specific RNA interference (RNAi) for AE2 was used to confirm whether AE2 could mediate the apoptosis induced by HG. Finally, the mechanisms of the AE2-mediated apoptosis were investigated by detecting mitochondrial permeability transition pore (mPTP, DeltaPsim) openings, reactive oxygen species (ROS) levels and Caspase-3 activity. We found that HG upregulated the AE2 expression and activity, increased [Cl(-)]i and induced the apoptosis in a time- and concentration-dependent manner. The apoptosis of HUVECs by HG was possibly mediated by AE2 through an mPTP-ROS-Caspase-3 dependent pathway. These findings suggested that AE2 was likely to be a glucose-sensitive transmembrane transporter and a novel potential therapeutic target for diabetic vasculopathy.

Hydrogen Sulfide Protects Against Ischemia-Reperfusion Injury in an In Vitro Model of Cutaneous Tissue Transplantation

Ischemia-reperfusion injury (IRI) is a source of morbidity and mortality in many clinical scenarios, and has as one of its many consequences the induction of cellular apoptosis. Hydrogen sulfide (H2S) may decrease cellular metabolism in a reversible, nontoxic manner. An in vitro model of cutaneous tissue transplantation was developed to assess whether H2S could ameliorate cellular injury caused by IRI. Human umbilical vein endothelial cells (HUVECs) were treated with media containing NaHS (0, 10 microM, 100 microM, or 1 mM) and exposed to normoxia (21% oxygen), hypoxia (1%), or anoxia (0%). Cells were then returned to normoxia, and apoptosis was quantified using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Fibroblasts (3T3s) were treated with H2S and exposed to anoxia in a similar fashion. Treatment with H2S resulted in a significant decrease in apoptosis in HUVECs and 3T3s subjected to IRI. Toxicity of H2S was not observed, although the protective effect was less evident at higher doses. This is the first study to examine H2S and the cellular components of cutaneous flaps in the setting of IRI. Our results demonstrate that H2S significantly decreases apoptosis in vitro in the setting of IRI. These data suggest H2S may mitigate IRI in vivo, and, therefore, has potential as a therapy for improving tissue survivability in clinical scenarios.

Tormentic acid reduces vascular smooth muscle cell proliferation and survival

The triterpene tormentic acid (TA) has been reported to exhibit anticancer, anti-inflammatory and anti-atherogenic properties, and minimal toxicity has been detected in in vivo. Vascular smooth muscle cell (VSMC) proliferation and apoptosis resistance are hallmarks of vasculoproliferative diseases, such as post-angioplasty restenosis. The present study was designed to assess the effects of TA on the phenotype of cultured VSMC. The exposure of VSMC to TA (30 μM) significantly increased apoptosis of serum-deprived A7r5 cells, whereas cell survival in the presence of 10% fetal bovine serum was less affected by the drug. On the other hand, even in the presence of serum, A7r5 cell proliferation was significantly inhibited by TA, an effect that persisted for at least 8 days of daily administration of TA. As preservation of endothelial integrity is critical to normal vascular function, we also evaluated the effects of TA on human umbilical cord endothelial cells (HUVEC). Interestingly, TA did not produce significant changes in the levels of apoptosis and proliferation of HUVEC. Our data indicate that TA is a VSMC apoptosis inducer and proliferation inhibitor. The anti-growth action in VSMC in the presence of serum, and the absence of significant effects in endothelial cells suggest that TA may control VSMC abnormal proliferation and cell death resistance without affecting the normal vasculature. We conclude that TA should be investigated further as a potential tool for the prevention and treatment of proliferative vascular diseases, particularly in the setting of post-angioplasty restenosis.

Effect of estradiol, progesterone and testosterone on apoptosis- and proliferation-induced MAPK signaling in human umbilical vein endothelial cells

Sex hormones induce death or cell proliferation in various cell lines and in primary cultures. However, the signal transduction pathways involved in the regulation of proliferation and apoptosis in endothelial cells have not been fully elucidated. Here, we report that progesterone and testosterone induce apoptosis in HUVECs in a p38- and JNK-dependent manner, and that estradiol promotes proliferation via the activation of ERK2. We showed that, at physiological doses, progesterone and testosterone promoted p38, but not JNK, phosphorylation. Hormone inhibitors, on the other hand, prevented p38 phosphorylation. When supraphysiological doses were applied, both p38 and JNK were phosphorylated, causing apoptotic cell death. The addition of hormone inhibitors at an appropriate concentration did not prevent cell death or the phosphorylation of p38 and JNK. Estradiol, at physiological doses, promoted an increase in ERK2 phosphorylation that was blocked by fulvestrant. At physiological and supraphysiological doses, it promoted a proliferative effect. In conclusion, these findings suggest that JNK has an important pro-apoptotic function following progesterone and testosterone treatment in human endothelial cells, and that ERK2 has a proliferative effect following estradiol treatment.

Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array

BackgroundThe balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis.ResultsHuman umbilical vein endothelial cells (HUVECs) were treated with cobalt chloride (CoCl2) both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 μM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing.ConclusionThe exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs under mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulation in endothelial cells under physiological and pathological conditions.

Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway

Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K + from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.

Abstract 4010: Soluble TRAIL in Peripheral Blood is a Novel Marker for Acute Coronary Syndromes

Background: Apoptosis of vascular smooth muscle cells and endothelial cells in the fibrous cap is prone to plaque vulnerability. Recently, we found that CD4 T cells trigger the TRAIL/TRAIL receptor 2 (DR5) pathway and cause VSMC apoptosis in acute coronary syndrome (ACS). We also showed that activated plasmacytoid dendritic cells (pDC) produce INFα through TRL9 and amplify cytotoxic CD4 T cell functions by enhancing TRAIL expression. In this study, we examined whether soluble TRAIL (sTRAIL) in peripheral blood shows changes in ACS that may serve as predictive marker of vulnerable patients. Methods and Results: The sTRAIL levels were measured by ELISA in 110 patients undergoing coronary angiography, including 90 ACS patients and 20 chest pain syndrome patients without apparent coronary atherosclerosis. Fresh CD4 T cells were isolated from the peripheral blood of patients. The sTRAIL levels were lower in ACS than in controls (P<0.001). CD4 T cells capable of inducing HUVEC apoptosis expressed high levels of TRAIL in ACS patients (P<0.03), and negatively correlated with the sTRAIL levels (R=0.372, P<0.02). To examine whether sTRAIL levels could be used as marker of vulnerable patients with ACS, we measured sTRAIL levels in the time course and compared them with CPK and TnT after onset of ACS. While CPK and TnT of acute myocardial infarction elevated in the acute phase and recovered after 3days, sTRAIL showed negative parallel changes. Meanwhile, sTRAIL showed the same changes in unstable angina without CPK and TnT elevation. The levels of sTRAIL more decreased in patients with multiple coronary artery stenosis than in that without coronary artery stenosis while the levels of hsCRP did not. Furthermore, given a cutoff value of 48.6pg/ml, sTRAIL was able to discriminate ACS from control groups with sensitivity of 62.4%, specificity of 85.0%, and area under the receiver-operating characteristic curve (AUC) of 0.736. In contrast, an hsCRP cutoff value of 1.25mg/l showed lower sensitivity (47.3%), specificity (77.8%), and AUC (0.597) for the diagnosis of ACS. Conclusions: Monitoring of TRAIL levels in ACS patients with both acute myocardial infarction and unstable angina was found useful in predicting vulnerability in patients with vulnerable plaques.

Endothelium-specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice

Hazardous environmental and genetic factors can damage endothelial cells to induce atherosclerotic vascular disease. Recent studies suggest that class III deacetylase SIRT1 may promote cell survival via novel antioxidative mechanisms. The current study tested the hypothesis that SIRT1, specifically overexpressed in the endothelium, is atheroprotective. Human umbilical vein endothelial cells (HUVECs) were used to study the effects of oxidized low-density lipoprotein (LDL) on SIRT1 expression. Endothelial cell-specific SIRT1 transgenic (SIRT1-Tg) mice were used to study the effects of SIRT1 on aortic vascular tone. SIRT1-Tg mice were crossed with apolipoprotein E null (apoE(-/-)) mice to obtain SIRT1-Tg/apoE(-/-) mice for the analysis of atherogenesis in the presence of endothelial overexpression of SIRT1. SIRT1 expression in HUVECs was increased by the treatment with oxidative LDL. Adenoviral-mediated overexpression of SIRT1 was protective of apoptosis of HUVECs. Calorie restriction increased, whereas high-fat diet decreased, the SIRT1 expression in mouse aortas. In SIRT1-Tg mice, high fat-induced impairment in endothelium-dependent vasorelaxation was improved compared with that of wild-type littermates. This was accompanied by an upregualtion of aortic endothelial nitric oxide synthase expression in the SIRT1-Tg mice. The SIRT1-Tg/apoE(-/-) mice had less atherosclerotic lesions compared with apoE(-/-) controls, without affecting blood lipids and glucose levels. These results suggest that endothelium-specific SIRT1 overexpression likely suppresses atherogenesis via improving endothelial cell survival and function.

Anxiolytic activity of Atrial Natriuretic Peptide and ß-Endorphin during acute Ethanol withdrawal in mice

Tumor angiogenesis and PI3K/Akt/mTOR pathway are two major molecular objectives for the treatment and management of breast cancer. Here we first time report the molecular mechanism of a marine sponge alkaloid derivative 4-chloro fascapysin (4-CF) for its anticancer and antiangiogenesis potential. It simultaneously targets multiple cancer and angiogenesis dynamics, such as proliferation, chemotaxis cell migration, and invasion, growth factors signaling cascade, autophagy and apoptosis in HUVEC and MDAMB-231 breast cancer cells. It inhibited the VEGF mediated microvessel sprouting and blood vessel formation in the matrigel plug of C57/BL6J mice. It inhibits the tumor growth in ET (solid) mouse tumor model. It significantly inhibited cell survival through PI3K/Akt/mTOR pathway, with attendant effects on key pro-angiogenesis factors like HIF-1α, eNOS and MMP-2/9. The cytotoxicity of 4-CF was reversed by co-treatment with the VEGF and Akt inhibitors sunitinib and perifosine, respectively or by the addition of neutralizing VEGF antibodies. The apoptotic potential of 4-CF was through mitochondrial dependent as illustrated through loss of mitochondrial membrane potential. The safety profile of 4-CF was acceptable as it exhibits five times high cytotoxic IC50 value in normal cells as well as no apparent toxicities in experimental tumor mice at therapeutic doses.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-III SVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24 kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24 h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Heme oxygenase‐1 improves the survival of discordant cardiac xenograft through its anti‐inflammatory and anti‐apoptotic effects

HO-1 is a rate-limiting enzyme in hemoglobin metabolism, and exerts anti-inflammatory as well as anti-apoptotic effects. Previous studies have shown that expression of HO-1 can prolong the survival of concordant transplanted organs. However, little is known about the precise effect and mechanism of HO-1 in discordant xenotransplantation. In this study, we investigated the role of HO-1 in discordant cardiac xenotransplantation. First, HUVECs were used to assess the effect of HO-1 on TNF-alpha-induced apoptosis. Results showed that TNF-alpha induced apoptosis of HUVECs in a dose-dependent manner. Moreover, induction of HO-1 by hemin suppressed TNF-alpha-induced apoptosis. However, the anti-apoptotic action of HO-1 was reversed by SnPP. The up-regulation of HO-1 by hemin treatment significantly prolonged the survival time of discordant cardiac xenograft, greatly reduced the swelling and apoptosis of myocardial cells, interstitial edema, lymphocyte infiltration, and thrombus formation in small vessels. Furthermore, HO-1 overexpression significantly attenuated the serum level of xenoantibody IgM, tissue deposition of IgM and complement 3 (C(3)) in endangium. Finally, HO-1 mitigated CD40L transcription in the xenograft and recipient spleen. These results indicate that the up-regulation of HO-1 can improve the survival of discordant cardiac xenograft by inhibiting apoptosis and alleviating inflammation and thrombosis.

Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of Src activity is associated with the inability of Src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and angiogenesis-restricted tumor dormancy. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. The most conclusive anti-angiogenic activity of AZM475271 was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor-induced neovascularization in response to systemic administration of AZM475271. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signaling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF - dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of Src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms.

Rosuvastatin stimulates eNOS and inhibits apoptosis in HUVECs exposed to sera from cardiovascular diseases patients

Rosuvastatin stimulates eNOS and inhibits apoptosis in HUVECs exposed to sera from cardiovascular diseases patients

Hypoxia induces apoptosis of HUVECs in an in vitro capillary model by activating proapoptotic signal p38 through suppression of ERK1/2

We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.

Design, synthesis, and preliminary biological evaluation of 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives

We synthesized a series of novel small molecules, 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives, by tandem reduction-oxirane opening of 2-nitroaroxymethyloxiranes in moderate or excellent yields. We investigated the effects of all of the compounds on HUVEC apoptosis and A549 cell growth. The results showed that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine was the most effective small molecule in promoting HUVEC apoptosis and inhibiting A549 cell proliferation, but 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine could remarkably inhibit HUVEC apoptosis and might induce the formation of microvessel.

Docosahexaenoic acid induces ciap1 mRNA and protects human endothelial cells from stress-induced apoptosis

Induction of apoptosis represents a potential reaction of endothelial cells (ECs) after injury of the vascular endothelium. Beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in vascular diseases are widely recognized although the responsible mechanisms are not fully understood. Because it is not known whether PUFAs modulate EC apoptosis, we investigated the effects of n-3 and n-6 PUFAs on 4-hydroxynonenal (HNE)-induced EC apoptosis by annexin V staining and caspase-3 activation assays. Pretreatment with the n-3 fatty acid docosahexaenoic acid (DHA) reduced HNE-induced EC apoptosis. DHA-treated cells did not show the pronounced drop in intracellular GSH after HNE exposure seen in vehicle- or n-6 arachidonic acid-treated cells. This is most likely due to increased GSH levels in DHA-treated cells. Furthermore, DHA pretreatment increased ciap1 mRNA levels and transfection of cIAP1 small interfering RNA abolished the protective effect of DHA in HNE-induced apoptosis in HUVECs. Thus pretreatment of HUVECs with DHA reduces HNE-induced oxidative stress and apoptosis, and the protective effects of DHA seem to be dependent on cIAP1. The results provide a possible new mechanism for the atheroprotective effects of n-3 fatty acids in vascular disease.

Interferon γ–Inducible Protein 10 Selectively Inhibits Proliferation and Induces Apoptosis in Endothelial Cells

Interferon gamma-inducible protein 10 (IP-10) has antitumor effects in various murine models. The IP-10 receptor has two distinct splice variants, CXCR3A and CXCR3B, that have paradoxical effects after ligand-receptor interaction. To characterize the putative antiangiogenic effects of IP-10, we measured proliferation rates and apoptosis in human umbilical vein endothelial cells (HUVECs), fibroblasts, and A375 melanoma or WIDR adenocarcinoma cell lines after exposure to the recombinant protein. CXCR3A (activating) and CXCR3B (inhibitory/proapoptotic) messenger RNA (mRNA) expression levels in fibroblasts, 2 human tumor cell lines, T lymphocytes, and HUVECs of varying cell densities were characterized. IP-10 resulted in dose-dependent and selective inhibition of proliferation and countered the proliferative effects of vascular endothelial growth factor in HUVECs but did not affect fibroblasts or 2 human tumor cell lines. In addition, IP-10 resulted in potent and selective induction of apoptosis in HUVECS but had no effect on fibroblasts or A375 melanoma. Confluent HUVECs had a predominance of mRNA for the CXCR3B splice variant by reverse transcriptase-polymerase chain reaction, and the ratio of CXCR3B to CXCR3A mRNA was >40 in HUVECs, compared with </=10 in the other cell types. Moreover, CXCR3B mRNA levels were significantly higher in proliferating compared with confluent HUVECs. In vivo, systemic IP-10 administration resulted in slower A375 xenograft growth rates compared with control-treated animals, and immunohistochemical staining showed decreased microvessel density in xenografts of IP-10-treated mice. IP-10 has antiangiogenic properties and selective effects on endothelial tissue that may be secondary to higher levels of the CXCR3B inhibitory/proapoptotic receptor in that cell type, particularly in its actively proliferating state.

β-Carotene and angiogenesis

Abstract Carotenoids and retinoids modulate growth and differentiation of a variety of cell types and are fundamental regulators of development. Endothelial cells play an important role in angiogenesis, which is essential for organogenesis and tissue remodeling, but also inflammatory response or carcinogenesis. Binding to the retinoid (RARs) or rexinoid (RXRs) receptors, all-trans-RA, 13-cis-RA, 9-cis-RA, and synthetic retinoids and rexinoids showed antiangiogenic properties in several models. However, the role of β-carotene in endothelial cell function and angiogenesis is still poorly characterized. Although in our experiments, β-carotene used in nontoxic concentrations (up to 3 μM) had no detectable effect on the proliferation or apoptosis of HUVECs or umbilical-cord-blood-derived endothelial progenitors; β-carotene did not change the tubulogenic activity of cells in an in vitro angiogenesis model, but it potently activated the migration of endothelial and progenitor cells. β-Carotene also promoted the development of microcapillaries in a matrigel plug injected subcutaneously into mice. The analysis of microarray data from endothelial cells revealed that β-carotene modified the expression of genes involved in activation of chemotaxis, cell/cell and cell/matrix adhesion, matrix reorganization, G-protein-regulated intracellular signaling as well as genes involved in the rapid remodeling of the actin cytoskeleton. We conclude that physiological levels of β-carotene stimulate early steps of angiogenic activity of endothelial cells by activation of cellular migration as well as matrix reorganization and reduction of cell adhesion.

The inhibition of angiogenesis by antisense oligonucleotides to clusterin

Angiogenesis is a primary disease target in ocular retinopathy and a secondary target in numerous other angiogenic diseases such as cancer, rheumatoid arthritis and psoriasis. Clinical trials using antiangiogenic antisense oligonucleotides (aso's) for the treatment of ocular disorders or cancer are well advanced. Clusterin aso's are currently under investigation for the treatment of prostate cancer. We have investigated the antiangiogenic properties of clusterin aso's using a capillary cell (HUVEC) viability assay. In this study we included aso's to known apoptosis modulators (bcl-2, bcl-xl and survivin) which were previously identified in HUVEC's. We have also studied the effect of clusterin aso's on angiogenesis using an in vitro, matrigel assay and on HUVEC apoptosis using an ELISA DNA fragmentation assay. Clusterin, bcl-2, bcl-xl and survivin aso's were all found to inhibit HUVEC growth. The apoptosis-inducing drugs paclitaxel, camptothecin and doxorubicin were also found to inhibit HUVEC proliferation. Combinations of aso's with these drugs demonstrated a minor additive but not synergistic inhibitory effect on HUVEC proliferation. Clusterin aso's were found to strongly inhibit angiogenesis and induce high levels of apoptosis in HUVECs. In cancer cells the prosurvival protein clusterin may protect the cells from apoptosis-inducing agents so that the clusterin aso's may act as chemosensitization agents. These data demonstrate a strong antiangiogenic action of clusterin aso's, that is not necessarily related to any chemosensitization effect of this agent.

Loss of TM-Dependent PC-Activation Predisposes to Diabetic Nephropathy: Potential Role of Endothelial Apoptosis.

Increased levels of soluble thrombomodulin (TM) in patients with diabetes mellitus are considered a marker of endothelial damage. It is unknown whether the loss of endothelial TM-function contributes to the progression of vascular complications in diabetes mellitus. To address whether the loss of TM-dependent protein C (PC) activation contributes to diabetic complications such as diabetic nephropathy two animal models were employed: (1) TMPro mice, which have been previously described and carry a point mutation in the TM-gene (E404P), resulting in a loss of TM-dependent PC-activation, and (2) hPC mice, which carry a transgene resulting in the expression of a mutant “hyperactivatable” PC, which can be activated by thrombin in the absence of TM. The mutant PC could be captured from plasma samples of hPC mice and activated ex vivo by thrombin in the absence of TM. hPC mice had a prolonged bleeding time. Following induction of diabetes by streptozotocin TAT levels were increased in diabetic control (wild type) mice and to a larger extent in TMPro mice, but not in diabetic hPC mice. In comparison to diabetic control mice the kidney weight was increased in diabetic TMPro mice, but not in diabetic hPC mice. Albuminuria was increased in diabetic TMPro mice and reduced in diabetic hPC mice in comparison to diabetic control mice, indicating increased glomerular damage in TMPro mice and partial protection from glomerular damage in hPC mice. Consistently, using a histological score glomerular damage was more severe in diabetic TMPro mice in comparison to diabetic control mice, while diabetic hPC mice were protected. Preliminary data suggest that the observed changes are associated with increased apoptosis in glomeruli of diabetic TMPro mice. Using HUVECs we were able to establish that high glucose concentrations (30 mM) reduce TM-dependent PC activation. The reduced TM-dependent PC activation is associated with increased apoptosis. Glucose induced apoptosis in HUVECs is associated with an increased Bax/Bcl-2 ratio, increased translocation of Bax into mitochondria, and increased caspase-3 activation. Activated PC normalizes the Bax/Bcl-2 ratio, prevents translocation of Bax, and reduces caspase-3 activity. Further studies using TRAPs and inhibitory antibodies established that the antiapoptotic effect of aPC in glucose stressed endothelial cells is mediated through a Par-1 and EPCR-dependent mechanism. The current data strongly suggest that the loss of the endothelial TM-PC system is not just a marker of endothelial damage in diabetic patients, but rather contributes to the progression of diabetic vascular complications.