Human Microvascular Endothelial Cells Cells Research Articles

Evaluation of the Antimicrobial, Cytotoxic, and Physical Properties of Selected Nano-Complexes in Bovine Udder Inflammatory Pathogen Control.

Mastitis in dairy cows is a worldwide problem faced by dairy producers. Treatment mainly involves antibiotic therapy, however, due to widespread antibiotic resistance among bacteria, such treatments are no longer effective. For this reason, scientists are searching for new solutions to combat mastitis, which is caused by bacteria, fungi, and algae. One of the most promising solutions, nanotechnology, is attracting research due to its biocidal properties. The purpose of this research was to determine the biocidal properties of nanocomposites as a potential alternative to antibiotics in the control of mastitis, as well as to determine whether the use of nanoparticles and what concentration is safe for the breeder and the animal. In this study, the effects of Ag, Au, Cu, Fe, and Pt nanoparticles and their complexes were evaluated in relation to the survival of bacteria and fungi isolated from cattle diagnosed with mastitis, their physicochemical properties, and their toxicity to bovine and human mammary epithelial cells BME-UV1 and HMEC (human microvascular endothelial cells). Moreover, E. coli, S. aureus, C. albicans, and Prototheca sp. invasion was assessed using the alginate bead (bioprinted) model. The NPs were tested at concentrations of 25, 12.5, 6.25, 3.125, 1.56 mg/l for Au, Ag, Cu and Fe NPs, and 10, 5, 2.5, 1.25, 0.625 mg/l for Pt. With the exception of Fe and Pt, all exhibited biocidal properties against isolates, while the AgCu complex had the best effect. In addition, nanoparticles showed synergistic effects, while the low concentrations had no toxic effect on BME-UV1 and HMEC cells. Synergistic effects of nanoparticles and no toxicity to bovine and human cells might, in the future, be an effective alternative in the fight against microorganisms responsible for mastitis, and the implementation of research results in practice would reduce the percentage of dairy cows suffering from mastitis. The problem of increasing antibiotic resistance is posing a global threat to human's and animal's health, and requires comprehensive research to evaluate the potential use of nanoparticles - especially their complexes - as well as to determine whether nanoparticles are safe for the breeders and the animals. The conducted series of studies allows further consideration of the use of the obtained results in practice, creating a potentially new alternative to antibiotics in the treatment and prevention of mastitis in dairy cattle.

Cell-Type-Specific Modulation of Hydrogen Peroxide Cytotoxicity and 4-Hydroxynonenal Binding to Human Cellular Proteins In Vitro by Antioxidant Aloe vera Extract.

Although Aloe vera contains numerous bioactive components, the activity principles of widely used A. vera extracts are uncertain. Therefore, we analyzed the effects of genuine A. vera aqueous extract (AV) on human cells with respect to the effects of hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE). Fully developed A. vera leaves were harvested and analyzed for vitamin C, carotenoids, total soluble phenolic content, and antioxidant capacity. Furthermore, human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures were treated with AV extract for one hour after treatment with H2O2 or HNE. The cell number and viability were determined using Trypan Blue, and endogenous reactive oxygen species (ROS) production was determined by fluorescence, while intracellular HNE–protein adducts were measured for the first time ever by genuine cell-based HNE–His ELISA. The AV extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq/g fresh weight) and cell-type-specific influence on the cytotoxicity of H2O2, as well as on endogenous production of ROS and HNE–protein adducts induced by HNE treatment, while AV itself did not induce production of ROS or HNE–protein adducts at all. This study, for the first time, revealed the importance of HNE for the activity principles of AV. Since HMEC cells were the most sensitive to AV, the effects of AV on microvascular endothelia could be of particular importance for the activity principles of Aloe vera extracts.

Modulation of the cell growth by Aloe Vera extract and hydrogen peroxide

Reactive oxygen species (ROS), reactive nitrogen species, and their counterpart antioxidant agents are essential for physiological signaling, while imbalances between oxidants and antioxidants may provoke pathological reactions causing a range of diseases including cancer. In addition, ROS can stimulate signal transduction pathways and lead to activation of key transcription factors such as Nrf2 and NF-κB modulating cell growth and redox balance [1]. The well-known medical plant Aloe vera contains an abundance of antioxidants, which are capable of neutralising ROS [2]. This work evaluates the effects of the genuine Aloe vera extract (AV) on human cells in vitro in respect to the toxic effects of hydrogen peroxide. After propagating Aloe vera for 1 year in 0.5 kg plastic pots, the first fully developed leaf, fourth from the top, was harvested and subjected to biochemical tests for determination of vitamin C, carotenoids and total soluble phenolic content, while total antioxidant capacity of the plant extract was analysed using Brand-Williams method [3]. Afterwards, four different human cell lines, notably HeLa (human cervical cancer), HMEC (human microvascular endothelial cells), HaCat (human keratinocytes) and HOS (human osteosarcoma) were treated with 2 concentrations of AV for 1 hour after one hour pre-treatment with ranging concentrations of H2O2, thus inducing oxidative stress. The viability of the cells was determined by an MTT-based vitality assay, EZ4U (Biomedica, Vienna, Austria). The plant extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq./g FW), mostly likely due to the combined effects of its antioxidants ingredients. However, while H2O2 inhibited in a concentration dependent manner the growth of all cell lines except HOS, the extract of AV did not show any particular effects except a slight stimulation of the HeLa and HMEC cells. We may assume that antioxidants of the plant extract are not involved in the observed growth modifying effects on the different cell lines used. More likely, some components of the plant extract could interfere with the cellular antioxidants and redox signalling and its effects together with hydrogen peroxide. The above actions of AV confirmed that it can potentially act as relatively safe natural source of medical remedies since there were no toxic effects observed, while further studies on the mechanisms of its actions are needed.

Metformin exhibits radiation countermeasures efficacy when used alone or in combination with sulfhydryl containing drugs.

Metformin, a biguanide drug used in the treatment of type II diabetes, was evaluated alone and in combination with amifostine, captopril, MESNA or N-acetyl-cysteine (NAC) for its ability to protect when administered 24 h after irradiation. Mouse embryo fibroblasts (MEF), human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were exposed to 4 Gy in vitro. C3H mice were exposed to 7 Gy and evaluated utilizing an endogenous spleen colony assay system. Amifostine and WR1065, administered 30 min prior to irradiation, were used as positive controls. Treatment of MEF, HMEC and SA-NH cells with metformin elevated survival levels by 1.4-, 1.5- and 1.3-fold compared to 1.9-, 1.8- and 1.6-fold for these same cells treated with WR1065, respectively. Metformin (250 mg/kg) was effective in protecting splenic cells from a 7 Gy dose in vivo (protection factor = 1.8). Amifostine (400 mg/kg), administered 30 min prior to irradiation resulted in a 2.6-fold survival elevation, while metformin administered 24 h after irradiation in combination with NAC (400 mg/kg), MESNA (300 mg/kg) or captopril (200 mg/kg) enhanced survival by 2.6-, 2.8- and 2.4-fold, respectively. Each of these agents has been approved by the FDA for human use and each has a well characterized human safety profile. Metformin alone or in combination with selected sulfhydryl agents possesses radioprotective properties when administered 24 h after radiation exposure comparable to that observed for amifostine administered 30 min prior to irradiation making it a potentially useful agent for radiation countermeasures use.

SR16388: a steroidal antiangiogenic agent with potent inhibitory effect on tumor growth in vivo

Angiogenesis is one of the major processes controlling growth and metastasis of tumors. Angiogenesis inhibitors have been targeted for the treatment of various cancers for more than 2 decades. We have developed a novel class of steroidal compounds aimed at blocking the angiogenic process in cancerous tissues. Our lead compound, SR16388, is a potent antiangiogenic agent with binding affinity to estrogen receptor-α (ER-α) and -β (ER-β) at the nanomolar range. This compound inhibited the proliferation of human microvascular endothelial cells (HMVEC) and various types of human cancer cells in vitro. SR16388 inhibited embryonic angiogenesis as measured in the chick chorioallantoic membrane (CAM) assay. The blood vessel density in the CAM was greatly reduced after the embryos were treated with 3μg/CAM of SR16388 for 24h. SR16388 at a dose of 2μM prevented tube formation in Matrigel after HMVEC cells were treated for 8h. In a modified Boyden chamber assay, SR16388 inhibited the migration of HMVECs by 80% at 500nM. Using a novel in vivo Fibrin Z-chamber model, we demonstrated that SR16388 at a single daily oral dose of 3mg/kg for 12 days significantly inhibited the granulation tissue (GT) thickness and the microvessel density of the GT as compared to control. More importantly, SR16388 down-regulated the pro-angiogenic transcription factors, hypoxia inducible factor 1α (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) in non-small cell lung cancer (NSCLC) cells. Together, these effects of SR16388 can lead to the reduction of vascularization and tumor growth in vivo.

Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC-BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.

Human microvasculature fabrication using thermal inkjet printing technology

The current tissue engineering paradigm is that successfully engineered thick tissues must include vasculature. As biological approaches alone, such as VEGF, have fallen short of their promises, one may look for an engineering approach to build microvasculature. Layer-by-layer approaches for customized fabrication of cell/scaffold constructs have shown some potential in building complex 3D structures. With the advent of cell printing, one may be able to build precise human microvasculature with suitable bio-ink. Human microvascular endothelial cells (HMVEC) and fibrin were studied as bio-ink for microvasculature construction. Endothelial cells are the only cells to compose the human capillaries and also form the entire inner lining of cardiovascular system. Fibrin has been already widely recognized as tissue engineering scaffold for vasculature and other cells, including skeleton/smooth muscle cells and chondrocytes. In our study, we precisely fabricated micron-sized fibrin channels using a drop-on-demand polymerization. This printing technique uses aqueous processes that have been shown to induce little, if any, damage to cells. When printing HMVEC cells in conjunction with the fibrin, we found the cells aligned themselves inside the channels and proliferated to form confluent linings. The 3D tubular structure was also found in the printed patterns. We conclude that a combined simultaneous cell and scaffold printing can promote HMVEC proliferation and microvasculature formation.

582 POSTER An acquired point mutation in MEK2 causes resistance to allosteric MEK inhibitors

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 μM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.

Effects of defibrotide on tumour adhesion and invasion in multiple myeloma, breast cancer and renal cell cancer

8600 Background: Defibrotide (DF), an polydisperse oligonucleotide, has been shown to increase in vivo sensitivity of multiple myeloma (MM) cells to conventional chemotherapy. The tumor microenvironment can influence drug response, resulting in chemoresistance, by interactions between cell surface integrins and extracellular matrix (ECM) proteins. Heparanase (HPSE) is an endoglycosidase involved in degradation and remodeling of the ECM. We investigated if the expression of adhesion molecules in human microvascular endothelial cells (HMEC) is modified by coculture with MM cells and if DF regulates the expression of these molecules. We also evaluated if DF modulates adhesion of MM cells to fibronectin. Finally, we evaluated if DF regulates the expression of HPSE in human breast carcinoma and renal adenocarcinoma (RCC) cell lines. Methods: HMEC were co-cultured with MM cells for 48h with and without DF. The expression of ICAM-1 and E-selectin in HMEC and MM cells was examined by RT-PCR and by FACS. HPSE gene expression was evaluated in RCC and breast carcinoma cells treated with DF or saline. Adhesion of MM cells to fibronectin was evaluated in 96 well plates coated with fibronectin with and without DF for 24 h. Attached MM cells were detected by calcein staining. Results: Coculture with MM cells substantially induced the expression of ICAM-1 and E-selectin (p<0.01) in HMEC cells. DF significantly downregulated ICAM-1 and E- selectin gene expression in HMEC co-cultured with MM cells (p<0.01). Furthermore, DF significantly downregulated HPSE gene expression in the RCC and breast carcinoma cell lines (p<0.01). Adhesion of MM cells to fibronectin was significantly reduced by DF (p<0.01). Conclusions: Our results demonstrate that DF downregulates the expression of adhesion molecules in MM and activated endothelial cells. DF significantly inhibited adhesion of MM cells to fibronectin. Theses findings, together with the downregulation of HPSE in human cancer cell lines, suggest that the anti-cancer activity of DF is due to its combined effects on tumour cell interactions with their microenvironment and provide a rational framework for future studies of DF in combination with other agents for the treatment of MM and other neoplasias, including breast cancer and RCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Gentium S.p.A. Gentium S.p.A.

Maintenance of Manganese Superoxide Dismutase (SOD2)-Mediated Delayed Radioprotection Induced by Repeated Administration of the Free Thiol Form of Amifostine

Thiol-containing drugs such as WR1065, the free thiol form of amifostine, have been shown to induce a delayed radioprotective effect in both malignant and non-malignant cells. In mammalian cells exposed to a dose as low as 40 microM WR1065, the redox-sensitive nuclear transcription factor kappaB (NFkappaB) is activated, leading to an elevation in the expression of the antioxidant gene manganese superoxide dismutase (SOD2) and a concomitant increase in active SOD2 enzyme levels that peaks 24 to 32 h later. Exposure of cells to ionizing radiation during the period of elevated SOD2 enzymatic activity results in an enhanced radiation resistance. This is seen as an increase in surviving fraction as determined by standard colony formation assays. To determine whether this delayed radioprotection can be maintained over a prolonged period in cells of either malignant or non-malignant origin, both human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were grown to confluence and exposed to 40 muM WR1065 using three administration protocols: (1) daily drug exposure for 10 days followed each day by irradiation with 2 Gy; (2) drug exposure once every 48 h followed by irradiation with 2 Gy 48 h later for 14 days; and (3) drug exposure every 72 h followed by irradiation with 2 Gy 72 h later for 12 days. As a function of each experimental condition, cell numbers and associated SOD2 enzymatic activities were measured at the time of each irradiation. None of the treatment conditions were toxic to either HMEC or SA-NH cells. SOD2 activity was elevated 5.3- and 1.8-fold over background on average for HMEC exposed to 40 microM WR1065 every 24 or 48 h, respectively. Likewise, SOD2 activity was elevated in SA-NH mouse sarcoma cells 7.8- and 4.9-fold after daily exposure to WR1065 or exposure to WR1065 once every 48 h, respectively. Both HMEC and SA-NH cells exhibited enhanced radiation resistance that correlated with the increase in SOD2 activity. The average respective increases in cell survival were 1.33 +/- 0.01 (SEM), 1.23 +/- 0.01 and 1.04 +/- 0.01 for HMEC exposed to WR1065 every 24, 48 and 72 h, respectively, and 1.27 +/- 0.01, 1.18 +/- 0.02 and 1.02 +/- 0.02 for SA-NH cells exposed to WR1065 every 24, 48 and 72 h, respectively. Both the elevation in WR1065-induced SOD2 enzymatic activity and the corresponding increase in radiation resistance were completely inhibited in HMEC and SA-NH cells transfected with human or mouse SOD2 siRNA oligomers and irradiated 24 h later. These data demonstrate that a delayed radioprotective effect can be induced and maintained over a prolonged period in both non-malignant and malignant cells exposed to thiol-containing drugs such as WR1065. For non-malignant cells this represents a novel paradigm for radiation protection. The ability of WR1065 to induce a persistent elevated radiation resistance in malignant cells, however, suggests a new potential concern regarding the issue of tumor protection in patients exposed to thiol-containing drugs.

1‐Oleoyl‐2‐acetyl‐glycerol (OAG) dependent Ca2+ influx is inhibited by dominant negative TRPC6 in human microvascular endothelial cells

Ca2+ signaling in endothelial cells plays a key role in maintaining the barrier function between blood vessel and tissues. The canonical transient receptor potential (TRPC) ion channel are considered important in Ca2+ signal generation, but their roles in human microvascular endothelial cells (HMVECs) remain elusive. We previously showed that VEGF, acting through DAG could increase cation entry into HMVECs through a store independent process consistent with TRPC6 activation. To assess the response of HMVECs expressing endogenous, overexpressed and dominant‐negative TRPC6 channels to OAG, a structural analog of DAG, VEGF and other agents, adenoviruses were generated that encoded the DN‐TRPC6 or WT TRPC6 cDNAs (courtesy of Wiloliam Cole, University of Calgary). Calcium concentration was assessed in 20μM Fura‐2AM‐loaded HMVEC. Fluorescence intensity of the dye at 340nm excitation to that at 380nm (Rnorm) was measured. OAG (200uM) induced an increase in Rnorm ‐ indicating increased calcium ‐ in control transduced and WT TRPC6, but the OAG mediated increase in calcium was blocked by the dominant negative isoform of TRPC6 adenovirus. (p&lt;0.05, Student Newman Keuls Post‐hoc ANOVA). In HMVEC cells, natively expressed TRPC6s are involved in DAG mediated calcium entry. DN‐TRPC6 may block this calcium entry either through inhibition of TRPC6 homodimers, or by blocking functional TRPC heterodimers. The effect of VEGF on DN‐TRPC6 HMVECs will also be presented.

Simultaneous Deposition of Human Microvascular Endothelial Cells and Biomaterials for Human Microvasculature Fabrication Using Inkjet Printing

The current tissue engineering paradigm is that successfully engineered thick tissues must include vasculature. As biological approaches alone such as VGEF have fallen short of their promises, one may look for an engineering approach to build microvasculature. Layer-by-layer approach for customized fabrication of cell/scaffold constructs have shown some potential in building complex 3D structures. With the advent of cell printing, one may be able to build precise human microvasculature with suitable bioink. Human Microvascular Endothelial Cells (HMEC) and fibrin were studied as bioink for microvasculature construction. Endothelial cells are the only cells to compose the human capillaries and also the major cells of blood vessel intima layer. Fibrin has been already widely recognized as tissue engineering scaffold for vasculature and other cells, including skeleton/smooth muscle cells and chondrocytes. In our study, we precisely fabricated micron-sized fibrin channels using a drop-on-demand polymerization. This printing technique uses aqueous processes that have been shown to induce little, if any, damage to cells. When printing HMEC cells in conjunction with the fibrin, we found the cells aligned themselves inside the channels and proliferated to form confluent linings. Current studies to characterize the biology and functionality of these engineered microvascular structures will be presented. The preliminary data suggests that a combined simultaneous cell and scaffold printing can promote HMEC proliferation and microvasculature formation.

Co-Culture of Myeloma and Endothelial Cells: Defibrotide Downregulates Heparanase and Pro-Angiogenic Growth Factors.

Introduction: Heparanase (HPSE) expression in humans has been associated with advanced progression and metastasis of many tumor types, including multiple myeloma (MM), where its activity is correlated with altered gene expression that may promote an aggressive tumor phenotype with high microvessel density (ref). These findings indicate an important role of HPSE in regulating metastasis, angiogenesis and progression of MM. Defibrotide (DF) is an orally bio-available polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic, anti-adhesive and anti-angiogenic properties. Recently, we have shown that DF is able to downregulate HPSE gene expression and activity in MM cell lines (International myeloma workshop, Greece, 2007).Methods: We investigated whether the expression of HPSE and angiogenic growth factors (FGF-2 and VEGF) in human microvascular endothelial cells (HMEC) are modified by co-culture with MM cells or by growing in the media of MM cells. In addition, we evaluated whether DF has activity in regulating the expression of HPSE, FGF-2 and VEGF in HMEC co-cultured with MM cells. We then tested the effect of DF on the invasiveness of MM cells activated with HPSE. HMEC cells were co-cultured with RPMI 8226 MM cells for 48h in the presence and absence of DF (at dose of 150μg/ml). HMEC was also grown alone for 48h in MM cell-conditioned media. The expression of HPSE and angiogenic growth factors (FGF-2 and VEGF) present in endothelial cells were examined through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Tumor invasion was evaluated using the BD BioCoat MatrigelTM invasion system (BD Bioscience).Results: Coculture with RPMI 8226 cells substantially induced the expression of HPSE (7.2 fold) and angiogenic growth factors (3-5 fold) in HMEC compared with endothelial cells growing alone. DF was able to downregulate HPSE, FGF-2 and VEGF gene expression in HMEC co-cultured with RPMI 8226 cells (4.0; 6.0-8.0 fold, respectively). Surprisingly, addition of conditioned media in absence of MM cells resulted in 6.5 fold of elevation of HPSE but not growth factors expression in HMEC. These results show that components released by MM cells are sufficient to modulate HPSE. However, to alter growth factor expression, HMEC needed to be culture in the presence of RPMI 8226 cells. Additionally, HPSE increased the invasiveness of RPMI 8226 cells and DF was able to significantly decrease MM invasiveness in the Matrigel assay by 50% (p<0.05).Conclusion: Taken together, these results suggest that cross-talk between MM and endothelial cells leads to enhanced angiogenesis. HPSE is a key factor in this process, correlating with both angiogenic stimulus and MM progression. Moreover, DF is able to downregulate HSPE and growth factor expression in activated HMEC induced by MM, as well as reducing the invasiveness of MM in this system. These observations suggest a potent potential anti-tumor effect of DF and support further preclinical evaluation in MM models as well as ongoing clinical studies in this setting.

Defibrotide Counteracts the Modifications of Anti-Thrombotic Phenotype of Endothelial Cells Induced by Thalidomide.

Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro.Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p<0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p<0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p<0.01).Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

7-Ketocholesterol activates caspases-3/7, -8, and -12 in human microvascular endothelial cells in vitro

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 μM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.

Activation of caspase-9 with irradiation inhibits invasion and angiogenesis in SNB19 human glioma cells

Glioblastoma multiforme, the most common brain tumor, typically exhibits markedly increased angiogenesis, which is crucial for tumor growth and invasion. Antiangiogenic strategies based on disruption of the tumor microvasculature have proven effective for the treatment of experimental brain tumors. Here, we have overexpressed human caspase-9 by stable transfection in the SNB19 glioblastoma cell line, which normally expresses low levels of caspase-9. Our studies revealed that overexpression of caspase-9 coupled with radiation has a synergistic effect on the inhibition of glioma invasion as demonstrated by Matrigel assay (> 65%). Furthermore, sense caspase stable clones cocultured with fetal rat brain aggregates along with radiation showed complete inhibition as compared to the parental and vector controls. During in vitro angiogenesis, SNB19 cells cocultured with human microvascular endothelial cells (HMEC) showed vascular network formation after 48-72 h. In contrast, these capillary-like structures were inhibited when HMEC cells were cocultured with sense caspase stable SNB19 cells. This effect was further enhanced by radiation (5 Gy). Signaling mechanisms revealed that apoptosis is induced by cleavage of caspase-9 by radiation, loss of mitochondrial membrane potential and activation of caspase-3. These results demonstrate that activation of caspase-9 disrupts glioma cell invasion and angiogenesis in vitro. Hence, overexpression of proapoptotic molecules such as caspase-9 may be an important determinant of the therapeutic effect of radiation in cancer therapy.

Antiangiogenic potential of 10-hydroxycamptothecin

To investigate the antiangiogenic potential of 10-hydroxycamptothecin (HCPT), the proliferation of human microvascular endothelial cells (HMEC) and seven human tumor cell lines were detected by SRB assay, and the endothelial cell migration and tube formation were assessed using two in vitro model systems. Also, inhibition of angiogenesis was determined with a modification of the chick embryo chorioallantoic membrane (CAM) assay in vivo. Morphological assessment of apoptosis was performed by fluorescence microscope. HCPT 0.313–5 μmol.L −1 treatment resulted in a dose-dependent inhibition of proliferation, migration and tube formation in HMEC cells, and HCPT 6.25–25 nmol.egg −1 inhibited angiogenesis in CAM assay. HCPT 1.25–5 μmol.L −1 elicited typical morphological changes of apoptosis including condensed chromatin, nuclear fragmentation, and reduction in volume in HMEC cells. HCPT significantly inhibited angiogenesis both in vitro and in vivo at relatively low concentrations, and this effect was related with induction of apoptosis in HMEC cells. These results taken collectively suggest that HCPT may be a potent antiangiogenetic and cytotoxic drug and further investigation is warranted.