Anti-endothelial Effects Research Articles

Maintenance Therapy or Metronomic Treatment Replacement Intravenous Phase in Acute Lymphoblastic Leukemia During COVID-19 Pandemic: New Strategy

Coronavirus pandemic has prompted physicians and healthcare providers to change the treatment for cancer patients, especially those with acute lymphoblastic leukemia. During the Covid19 pandemic, oncologists around the world must choose the best strategy for these immunocompromised patients to reduce coronavirus infection and maximize patient survival. Conventional chemotherapy with intravenous formulation may cause severe neutropenia and cause fever and infection in various parts of the body. COVID-19 pneumonia may occur in 16% of patients undergoing chemotherapy. The infection also mimics the symptoms of CMV pneumonia or pulmonary aspergillosis and P.Carini's pneumonia. The first solution for oncology patients is simply to stop standard treatment or delay treatment to overcome the dangerous course of coronavirus, but this strategy is more specific to solid tumors. (1) However, this option is not suitable for acute leukemia, especially symptomatic patients who need immediate treatment to improve their quality of life and survival.It makes sense for these cancer patients to be tested repeatedly. However, it isn,t possible to perform serology and airway sampling methods for PCR during patient visits worldwide, especially in low- and middle-income countries. In addition, classical chemotherapy requires hospitalization in patients, and Howard has an intravenous and second cannula for infusion, which increases the risk of COVID 19 with pulmonary complications. In this paper, an oral chemotherapy formula could replace intravenous chemotherapy, and we will evaluate this theory in this paper.ALL chemotherapy protocols include two different stages, including pre-injection and maintenance injections, which mainly include oral chemotherapy agents. Maintenance chemotherapy is an important part of the treatment of acute lymphoblastic leukemia (ALL) in children to prevent recurrence by eliminating the minimum residual disease (MRD). ALL chemotherapy is based on the oral daily dose of 6-dose low-dose of mercaptopurine and weekly low-dose, monthly intravenous methotrexate, and intravenous vincristine for a period of 2 to 3 years. (2) At low doses, conventional chemotherapy is the same as metronomic chemotherapy with changes in the immune system and anti-angiogenic effects in ALL patients. (3) In molecular picture, elevated CEC and EMP levels in peripheral blood can be thought of as indicative of endothelial damage and vascular dysfunction. Circulation of endothelial cells (CEC), endothelial progenitor cells (EPC) and endothelial microfibers (EMP), which are environmental indicators of vascular endothelial integrity in peripheral blood cells, are effective in maintaining therapeutic maintenance. A decrease in these cellular biomarkers and an increase in THBS-1 levels indicate that maintenance will reduce the activity of the endothelium and increase its resting state. The mechanisms by which maintenance therapy uses this anti-endothelial effect are unclear, but several points can be made. The direct effect on endothelial cells has already been demonstrated for 3 anticancer drugs involved in ALL protocols. VEGF levels remain low and unchanged during treatment with maintenance. Therefore, it is unlikely that VEGF will play a key role in these settings . (4)

Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect.

Maintenance chemotherapy is an important part of the treatment of ALL in children. It relies on the long-term oral administration of daily low-dose mercaptopurin and weekly low-dose methotrexate. Although it has been used in the clinic for decades, its mechanisms of action remain unclear. Here, we investigated different angiogenic and immune biomarkers to gain insights into the mechanisms of action of maintenance therapy in children with ALL. We thus monitored circulating endothelial cells (CEC), endothelial progenitor cells (EPC) and endothelial microparticles (EMP), pro-angiogenic factors (VEGF, VEGFR-1 and Ang-2), anti-angiogenic factor thrombospondin-1 (THBS1) and regulatory T lymphocytes (Treg) in 47 children with ALL during the maintenance phase of their treatment (at treatment initiation and after 6, 12 and 18 months). We observed a statistically significant decrease in EPC and EMP counts throughout the maintenance phase associated with a significant increase in THBS1 levels. No significant change was detected in other angiogenic markers or in Treg numbers.The results presented here indicate that maintenance therapy in children with ALL exerts its antitumor activity at least in part through anti-angiogenic effects, similar to those induced by metronomic chemotherapy. Larger studies are now warranted to validate these findings and determine their clinical implications.

Purified IgGs from Type 2 Diabetes with Atrial Fibrillation Induce Intracellular Calcium Release in Cardiomyocytes through IP3 Pathway

Higher risk of developing life threatening cardiac arrhythmias, such as atrial fibrillation (AF) has been found in type 2 diabetes mellitus (T2DM), but the mechanism underlying the association is largely unknown. Our previous studies showed that circulating IgGs from certain advanced T2DM patients displayed anti-endothelial effects and often induced intracellular Ca release in endothelial cells. The present study is to test the hypothesis that circulating IgGs from T2DM patients with AF perturb intracellular Ca homeostasis in atrial cardiomyocytes which in turn trigger AF. Protein A-purified IgGs were obtained from a cohort of T2DM subjects with AF and subjects without AF or other cardiovascular complications. The data show that IgG (1 µg/mL) from T2DM AF patients caused acute intracellular Ca release in cultured mouse atrial cardiomyocytes HL-1 cells compared to control groups (p < 0.001). The IgG-mediated Ca release was insensitive to verapamil (20 µM), mibefradil (25 µM) or BTP-2 (5 µM), indicating that the Ca release is not through voltage-gated Ca channels or store-operated Ca entry. On the other hand, application of Xestospongin C (10 µM) or 2-APB (100 µM), two membrane-permeable IP3 receptor antagonists, or knockdown of IP3 receptor, significantly decreased Ca release stimulated by the IgGs from T2DM AF patients (p < 0.01). Taken together, our data suggest that circulating IgGs alter intracellular Ca homeostasis in cardiomyocytes through IP3 pathway in T2DM AF patients. Our studies may provide the mechanistic understanding on a causal role of circulating IgGs in T2DM patients with AF and other symptomatic cardiac conduction diseases. The IgGs may serve as a biomarker for identifying a subset of T2DM patients with increased risk for development of AF.

Novel cis-restricted β-lactam combretastatin A-4 analogues display anti-vascular and anti-metastatic properties in vitro

Combretastatin A-4 (CA-4) is a naturally occurring microtubular-destabilising agent that possesses potent anti-tumour and anti-vascular properties both in vitro and in vivo. Clinical trials to date indicate that its water-soluble prodrug, combretastatin A-4 phosphate (CA-4P), is well tolerated at therapeutically useful doses. However, the stilbenoid structure of CA-4, consisting of two phenyl rings linked by an ethylene bridge, renders the compound readily susceptible to isomerisation from its biologically active cis-conformation to its more thermodynamically stable but inactive trans-isomer. To circumvent this problem, we synthesised a series of cis-restricted CA-4 analogues. Replacement of the ethylene bridge with a 1,4-diaryl-2-azetidinone (β-lactam) ring provided a rigid scaffold thus preventing cis-trans isomerisation. We previously documented that these tubulin-depolymerising β-lactam compounds potently induced cell cycle arrest and apoptosis in a variety of cancerous cell lines (including those displaying multidrug resistance) and ex vivo patient samples, whilst exerting only minimal toxicity to normal cells. The purpose of this study was to elucidate the effect of the β-lactam compounds on both tumour vascularisation and tumour cell migration, two critical elements that occur during the growth and metastatic progression of tumours. We established that two representative β-lactam compounds, CA-104 and CA-432, exerted both anti-endothelial effects [G2/M arrest and apoptosis of primary human umbilical vein endothelial cells (HUVECs)] and anti-angiogenic effects [inhibition of HUVEC migration and differentiation and reduced vascular endothelial growth factor (VEGF) release from MDA-MB-231 breast adenocarcinoma cells]. In addition, we established that lead analogue, CA-432, abrogated the migration of MDA-MB-231 cells indicating an anti-metastatic function for these compounds. In summary, our results to date collectively indicate that these cis-restricted β-lactam CA-4 analogues may prove to be useful alternatives to CA-4 in the treatment of cancer but with the added advantage of improved stability of the cis-isomer.

EphA2 blockade enhances the anti-endothelial effect of radiation and inhibits irradiated tumor cell-induced migration of endothelial cells.

EphA2 tyrosine kinase plays an important role in tumor angiogenesis, but whether targeting this pathway can affect response to ionizing radiation (IR) remains unknown. We investigated, using a soluble EphA2-Fc chimera, whether EphA2 inhibition could sensitize A549 and MCF-7 tumor cells, as well as human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HDMEC), to IR. EphA2-Fc resulted in a greater response of endothelial cells (EC) to IR than either treatment alone. EphA2-Fc significantly increased apoptosis and decreased clonogenic survival, tube formation and migration in irradiated EC after stimulation with vascular endothelial growth factor (VEGF), without an affecting their proliferation. No difference in proliferation or survival was found in A549 and MCF-7 tumor cells. In a co-culture model, EphA2-Fc inhibited an irradiated A549 cell-induced increase in EC migration. VEGF supplementation, as well as condiotioned medium from irradiated A549 cells, phosphorylated EphA2 in EC. The latter was abrogated by EphA2-Fc. EC were most sensitive to a combination of EphA2 inhibition and radiotherapy. The induction of paracrine growth factors and activation of EphA2 in EC suggest a protective mechanism that tumors probably use to attenuate IR-induced antivascular effects. Our data justify further investigation to explore targeting EphA2 in tumor radiosensitivity in vivo.

Sorafenib enhances the in vitro anti-endothelial effects of low dose (metronomic) chemotherapy

Angiogenesis inhibitors may enhance the effects of low dose (metronomic) chemotherapy. However, there is a wide range of novel angiogenesis inhibitors which must be tested in combinations with oral chemotherapy agents to assess the anti-endothelial and anti-cancer effects. This preliminary testing is most suited to high throughput in vitro models, rather than clinical trials. We aimed to establish an in vitro model and test the anti-endothelial and anti-cancer effects of the multi-kinase inhibitor sorafenib when used as a single agent and in combination with oral chemotherapy agents used at low concentrations. Micro-vascular endothelial cells and 3 cancer cell lines were utilised and an extended treatment strategy (96 h) was employed in order to mimic a continuous low dose anti-angiogenic chemotherapy regimen. Sorafenib significantly enhanced the anti-endothelial effect of low dose etoposide, paclitaxel and temozolomide. Sorafenib also significantly enhanced the anti-cancer effect of low dose etoposide, paclitaxel and temozolomide in SK-MEL-2 melanoma cells, producing an additive effect on inhibition of cell growth in all cases. These combinations appear to be the most promising for in vivo pre-clinical studies, with a view to testing in melanoma patients as a continuous dosing strategy, due to the in vitro additive inhibitory effect on growth seen in both endothelial and cancer cells.

Sorafenib enhances the in vitro anti-endothelial effects of low dose (metronomic) chemotherapy

Angiogenesis inhibitors may enhance the effects of low dose (metronomic) chemotherapy. However, there is a wide range of novel angiogenesis inhibitors which must be tested in combinations with oral chemotherapy agents to assess the anti-endothelial and anti-cancer effects. This preliminary testing is most suited to high throughput in vitro models, rather than clinical trials. We aimed to establish an in vitro model and test the anti-endothelial and anti-cancer effects of the multi-kinase inhibitor sorafenib when used as a single agent and in combination with oral chemotherapy agents used at low concentrations. Micro-vascular endothelial cells and 3 cancer cell lines were utilised and an extended treatment strategy (96 h) was employed in order to mimic a continuous low dose anti-angiogenic chemotherapy regimen. Sorafenib significantly enhanced the anti-endothelial effect of low dose etoposide, paclitaxel and temozolomide. Sorafenib also significantly enhanced the anti-cancer effect of low dose etoposide, paclitaxel and temozolomide in SK-MEL-2 melanoma cells, producing an additive effect on inhibition of cell growth in all cases. These combinations appear to be the most promising for in vivo pre-clinical studies, with a view to testing in melanoma patients as a continuous dosing strategy, due to the in vitro additive inhibitory effect on growth seen in both endothelial and cancer cells.

Inhibition of Neuropilin-1 by RNA-Interference and its Angiostatic Potential in the Treatment of Hepatocellular Carcinoma

Neuropilin-1 (Nrp1) was recently described as a novel receptor for the pro-angiogenic molecule vascular endothelial growth factor (VEGF), indicating a role in tumor angiogenesis and tumor progression. Recent data confirm this assumption by demonstrating that some tumor and endothelial cells express Nrp1. Therefore, we wanted to investigate the potential role of Nrp1-knockdown on hepatoma and endothelial cell function in vitro and tumor growth in vivo. Nrp1 knockdown in SVEC4 - 10 and Hepa129 cells and its influence on signal transduction (MAPK pP38, pAKT, pERK1 / 2) was analyzed by Western blot. Effects on endothelial tube formation were assayed in an in vitro and in vivo matrigel assay. In vivo, effects of siRNA-Nrp1 were analyzed in a subcutaneous hepatoma model. To verify effects on endothelial and tumor cells in vivo, immunohistochemistry for proliferation, apoptosis and endothelial vessels was performed. LightCycler and Western blot analysis showed efficient inhibition of gene expression in SVEC4 - 10 and Hepa129 cells following siRNA-Nrp1 transfection. Signal transduction pathways were not influenced after siRNA-Nrp1 treatment compared to the controls. Endothelial tube formation was reduced by 59 % and 94 % in vitro and in vivo compared to controls, corresponding to reduced VCAM expression. Subcutaneous tumor growth was not influenced after siRNA treatment. Intratumoral proliferation was not altered after treatment with siRNA-Nrp1, whereas microvessel density and apoptosis were reduced after treatment with siRNA-Nrp1 compared to siRNA-Ctrl. In conclusion, inhibition of Nrp1 expression led to strong anti-endothelial effects, whereas tumor cells and tumor growth were not affected.

Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non-small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Pharmacokinetic-directed dosing of vandetanib and docetaxel in a mouse model of human squamous cell carcinoma.

Docetaxel, usually administered according to maximum tolerated dose (MTD), can inhibit endothelial cell proliferation at low nanomolar concentrations. Docetaxel may exert antiangiogenic effects if dosed so plasma levels are maintained at low nanomolar concentrations over a prolonged time. We evaluated metronomic and MTD-based dosing of docetaxel with and without vandetanib, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity, in a head and neck xenograft model. A murine physiologically based pharmacokinetic model was modified to predict docetaxel distribution following i.p. administration to design dosing regimens that target prespecified plasma concentrations, for antiendothelial effects (metronomic), or exposure, to mimic 30 mg/m2 (weekly/MTD) docetaxel in humans. Animals were treated for 28 days with 1 mg/kg/d (DTX1) or 6 mg/kg q4d (DTX6) docetaxel with or without vandetanib (15 mg/kg/d p.o.) in mice bearing UMSCC2 tumor xenografts. The DTX1 dosing scheme was adjusted to treatment for 10 days followed by 9 days off due to severe gastrointestinal toxicity. All treatment groups significantly reduced tumor volume, tumor proliferation (Ki-67), and tumor endothelial cell proliferation (Ki-67/von Willebrand factor) compared with control. Addition of vandetanib to docetaxel treatment significantly enhanced tumor growth inhibition over single-agent therapy. A positive correlation of tumor endothelial cell proliferation with tumor growth rates demonstrates vandetanib and docetaxel antiangiogenic effects. Due to the morbidity observed with DTX1 treatment, it is difficult to clearly ascertain if metronomic schedules will be effective for treatment. Docetaxel with vandetanib is effective in treating UMSCC2 xenografts at concentrations relevant to exposures in humans.

Mixed connective tissue disease: what is behind the curtain?

Although there is still an emotional debate over the existence of mixed connective tissue disease, the evidence from animal models suggests that anti-U1RNP antibodies, similar to other autoantibodies in other connective tissue diseases (such as antisynthetase, anticentromere, and antitopoisomerase), play a pathophysiological role in this disease. Despite an antiendothelial effect of anti-U1RNP antibodies, which is reminiscent of anticentromere antibodies, patients with high-titer autoantibodies to U1RNP in the absence of anti-Sm antibodies do not usually have or develop typical systemic sclerosis. Instead, their severe Raynaud's syndrome is commonly accompanied by arthritis, which can be erosive, and by swollen/puffy hands and myositis. Pulmonary arterial hypertension is the major life-threatening complication in these patients and regular screening for this condition is essential.

Spotlight

Cheng et al., pp.345–353 Prolonged exposure to estrogens can induce tumors in laboratory animals and has been associated with breast and uterine cancers in humans. The proliferative effect of estrogen on breast epithelium is part of the mechanism. In addition, recently surfaced evidence demonstrated that estrogen metabolites can cause DNA damage and mutations, an important step in tumor initiation. Oxidation of two catechol estrogen (CE) metabolites, 4-hydroxyestradiol and 4-hydroxyestrone, generates quinone intermediates. They can react with purine bases to form DNA adducts that generate highly mutagenic apurinic sites; CEs can also generate oxygen radicals and DNA double strand breaks (DSB). Cheng et al. examined whether breast tumorigenesis is linked to inherited variants in five genes that are involved in detoxifying pathways of estrogen metabolites. Women harboring a greater number of high-risk genotypes of CE-metabolizing genes faced an increased risk of breast cancer. This trend was more pronounced in women who had never been pregnant or older at their first full-term pregnancy and therefore at greater risk for breast cancer. Cheng also examined whether the relationship between cancer risk and the genotypic polymorphism of CE-metabolizing genes was modified by the genotypes of DSB repair genes. They found that a joint effect was significantly associated with breast cancer development. This study supports the hypotheses that increased estrogen exposure confers a higher risk of breast cancer, in part caused by DSB in DNA. Brennetot et al., pp.446–450 High-frequency microsatellite instability (MSI-H) is due to either insertion or deletion of repetitive sequences during DNA replication and failure of the mismatch repair system (MMR) to correct these errors. As a consequence, MMR-deficient tumors accumulate thousands of small mutations genome-wide. Almost all tumors from patients with human non-polyposis colorectal cancer (HNPCC) are MMR deficient, as are 10–15% of sporadic colorectal, gastric and endometrial cancers.2 Allelic profiles of BAT-26 and BAT-25 obtained by independent fluorescent PCR of these markers. MSI-H tumors differ from microsatellite-stable (MSS) tumors in molecular and in clinicopathological attributes. Generally, patients with MSI-H tumors are reported to have a better clinical outcome than patients with MMR-proficient tumors. Hence, screening of all human tumors where the MSI phenotype is frequent would not only allow the treatment to be custom-tailored but also assist in the diagnosis of ambiguous HNPCC cases. The present study demonstrates that together the markers BAT-25 and BAT-26, two mononucleotide repeats widely used as part of a larger set of markers, are sensitive enough to establish the MSI status of human tumors. Furthermore, they can detect instability when the tumor cell content of a sample is as low as 5–10%. This observation is very important in the context of proposed regular MSI screening for all colorectal, gastric and endometrial cancers. Not only might fewer markers than the routinely suggested panel of five suffice to reliably establish the microsatellite status of tumors, the labor-intensive and time-consuming process of enriching tumor cells in contaminated samples may only be required in a small subset of biopsies. Merchan et al., pp.490–498 Most angiogenesis inhibitors were developed intentionally to interrupt new blood vessel formation. In an unexpected twist, the antiangiogenic activity of several cytotoxic agents was discovered accidentally, among them the widely used chemotherapeutic paclitaxel. In addition to its well-known microtubule-interfering effect, paclitaxel inhibits endothelial cell (EC) migration, invasion, capillary tube formation and proliferation at low concentrations in vitro. In vivo, it significantly reduces microvessel density and decreases vascular endothelial growth factor (VEGF) production. While paclitaxel's effects on ECs are well studied and clinical trials investigating its antiangiogenic potential are underway, the underlying mechanism has not yet been elucidated. Based on the latest results by Merchan et al. it appears that an approximately fivefold increased ability of human ECs to accumulate the drug when compared to other cell types is responsible for the enhanced sensitivity of ECs. However, at the same time low concentrations of paclitaxel upregulate the expression of Cox-2, an enzyme also known for its angiogenesis-stimulating activity. Combined treatment with the specific Cox-2 inhibitor celecoxib resolved the undesirable dichotomy and resulted in more pronounced antiendothelial effects than applying either compound alone. These findings could be extended to a clinical setting, where four patients with prostate cancer or malignant melanoma were treated with prolonged intravenous infusions of paclitaxel and celecoxib. The potent antiangiogenic activity observed in plasma specimens suggests that a combination treatment may be an attractive strategy to capitalize on the intrinsic antiangiogenic and cytotoxic effects of each agent alone.

Inhibition of endothelial cell functions by novel potential cancer chemopreventive agents

Endothelial cells (EC) play a major role in tumor-induced neovascularization and bridge the gap between a microtumor and growth factors such as nutrients and oxygen supply required for expansion. Immortalized human microvascular endothelial cells (HMEC-1) were utilized to assess anti-endothelial effects of 10 novel potential cancer chemopreventive compounds from various sources that we have investigated previously in a human in vitro anti-angiogenic assay. These include the monoacylphloroglucinol isoaspidinol B, 1,2,5,7-tetrahydroxy-anthraquinone, peracetylated carnosic acid (PCA), isoxanthohumol, 2,2′,4′-trimethoxychalcone, 3′-bromo-2,4-dimethoxychalcone as well as four synthetic derivatives of lunularic acid, a bibenzyl found in mosses [Int. J. Cancer Prev. 1 (2004) 47]. EC proliferation was inhibited with half-maximal inhibitory concentrations from 0.3 to 49.6μM, whereas EC migration was affected by most compounds at sub-micromolar concentrations. PCA and the bibenzyl derivative EC 1004 potently prevented differentiation of HMEC-1 into tubule-like structures. Overall, our data indicate that inhibition of endothelial cell function contributes to various extents to the chemopreventive or anti-angiogenic potential of these lead compounds.

Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for k1-5-induced antiangiogenesis.

We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the alpha- and beta-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.

Selective anti-endothelial effects of protracted low-dose BAL-9504, a novel geranylgeranyl-transferase inhibitor

Because the anti-endothelial effects of agents such as interferon alpha, endostatin, and various chemotherapeutic drugs appear to be optimal after protracted, frequent dosing, both in vitro and in vivo, using low concentrations/doses of drugs, we tested the geranylgeranyl transferase inhibitor BAL-9504 (( E, E, E) [2-oxo-2-[[(3,7,11,15-tetramethyl-2,6,10,14 hexadecatetraenyl)-oxy]amino]ethyl] phosphonic acid) in this manner on different human cell types, including endothelial, breast cancer cells and fibroblasts. We found that human endothelial cells were preferentially affected with respect to inhibition of proliferation, induction of apoptosis, suppression of adhesion to fibronectin, and blockade of cell migration, by daily exposure to low concentrations of BAL-9504 for 6 days. These results may have implications in terms of both increasing anti-tumor efficacy, mediated by antiangiogenic mechanisms, and reducing toxic side effects.

Mechanisms of action of antimalarials in inflammation: induction of apoptosis in human endothelial cells.

Antimalarials are beneficial therapeutic agents in systemic lupus and rheumatoid arthritis. These autoimmune diseases have abnormally low apoptosis of inflammatory cells. Both disorders have an abnormal angiogenesis. In the present report, antimalarials were demonstrated to selectively increase apoptosis of HUVECs in vitro. A 24-h exposure to 50 or 150 microM of the drugs was associated with a significant loss of substrate-adherent cells. Chloroquine exhibited an inhibitory effect on HUVEC proliferation over 7 days. Programmed cell death in HUVECs rendered nonadherent by chloroquine was confirmed by the induction of DNA fragmentation in floating cells. Northern blot analysis revealed a rapidly increased expression of the bcl-x(s) gene without any change in the expression of the bcl-2 gene, indicating that HUVECs under chloroquine were undergoing apoptosis. The onset of the apoptotic cascade in HUVECs appeared shortly after the addition of chloroquine. The effect of chloroquine on apoptosis was distinct from acute cell lysis and was restricted to HUVECs. Antimalarials also induced IL-1alpha production. In parallel, chloroquine alone did not increase the expression of IL-6. Anti-IL-1alpha Ab or IL-1Ra only marginally reversed chloroquine-induced depression of proliferation for the low drug concentration, but not the massive cell death effect at and above 50 microM. Taken together, these data may indicate that antimalarials repress angiogenesis. The autocrine mechanism involving IL-1alpha accounts only for a minor fraction of the full antiendothelial effect of chloroquine, which is mainly dependent on apoptosis.