Thalidomide attenuates nitric oxide mediated angiogenesis by blocking migration of endothelial cells

BACKGROUND: Thalidomide is an immunomodulatory agent, which arrests angiogenesis. The mechanism of anti-angiogenic activity of thalidomide is not fully understood. As nitric oxide is involved in angiogenesis, we speculate a cross-talk between thalidomide and nitric oxide signaling pathway to define angiogenesis. The aim of present study is to understand the mechanistic aspects of thalidomide-mediated attenuation of angiogenesis induced by nitric oxide at the cellular level. METHODS: To study the cellular mechanism of thalidomide-mediated blocking of angiogenesis triggered by nitric oxide, we used two endothelial cell based models: 1) wound healing and 2) tube formation using ECV 304, an endothelial cell line. These cell-based models reflect pro-angiogenic events in vivo. We also studied the effects of thalidomide on nitric oxide mediated egg yolk angiogenesis. Thalidomide could block the formation of blood vessels both in absence and presence of nitric oxide. Thalidomide effects on migration of, and actin polymerization in, ECV 304 cells were studied at the single cell level using live cell imaging techniques and probes to detect nitric oxide. RESULTS: Results demonstrate that thalidomide blocks nitric oxide-mediated angiogenesis in egg yolk model and also reduces the number of tubes formed in endothelial cell monolayers. We also observed that thalidomide arrests wound healing in presence and absence of nitric oxide in a dose-dependent fashion. Additionally, thalidomide promotes actin polymerization and antagonizes the formation of membrane extensions triggered by nitric oxide in endothelial cells. Experiments targeting single tube structure with thalidomide, followed by nitric oxide treatment, show that the tube structures are insensitive to thalidomide and nitric oxide. These observations suggest that thalidomide interferes with nitric oxide-induced migration of endothelial cells at the initial phase of angiogenesis before cells co-ordinate themselves to form organized tubes in endothelial cells and thereby inhibits angiogenesis. CONCLUSION: Thalidomide exerts inhibitory effects on nitric oxide-mediated angiogenesis by altering sub-cellular actin polymerization pattern, which leads to inhibition of endothelial cell migration

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40% increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80% increase in vascular density and pillar number augmentation by 420%. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturatio

Chick Embryo Partial Ischemia Model: A New Approach to Study Ischemia Ex Vivo

Background: Ischemia is a pathophysiological condition due to blockade in blood supply to a specific tissue thus damaging the physiological activity of the tissue. Different in vivo models are presently available to study ischemia in heart and other tissues. However, no ex vivo ischemia model has been available to date for routine ischemia research and for faster screening of anti-ischemia drugs. In the present study, we took the opportunity to develop an ex vivo model of partial ischemia using the vascular bed of 4th day incubated chick embryo. Methodology/Principal Findings: Ischemia was created in chick embryo by ligating the right vitelline artery using sterile surgical suture. Hypoxia inducible factor- 1 alpha (HIF-1a), creatine phospho kinase-MB and reactive oxygen species in animal tissues and cells were measured to confirm ischemia in chick embryo. Additionally, ranolazine, N-acetyl cysteine and trimetazidine were administered as an anti-ischemic drug to validate the present model. Results from the present study depicted that blocking blood flow elevates HIF-1a, lipid peroxidation, peroxynitrite level in ischemic vessels while ranolazine administration partially attenuates ischemia driven HIF-1a expression. Endothelial cell incubated on ischemic blood vessels elucidated a higher level of HIF-1a expression with time while ranolazine treatment reduced HIF-1a in ischemic cells. Incubation of caprine heart strip on chick embryo ischemia model depicted an elevated creatine phospho kinase-MB activity under ischemic condition while histology of the treated heart sections evoked edema and disruption of myofibril structures. Conclusions/Significance: The present study concluded that chick embryo partial ischemia model can be used as a novel ex vivo model of ischemia. Therefore, the present model can be used parallel with the known in vivo ischemia models in understanding the mechanistic insight of ischemia development and in evaluating the activity of anti-ischemic drug.status: publishe

Profiling the patterns of miRNAs and transcriptome of human vasculature under micro-gravity: A template study using space science for solving health problems on the Earth

Microgravity (MG) offers a unique condition, which promotes cellular growth and functions. MG can yield three-dimensional tissue specimens mimicking natural growth and easier self-association of cells unlike traditional cultures. The vascular system is capable of remodeling its structure surprisingly in short time frame and adaptation to new environment. Treatment of endothelial monolayer and embryo vascular plexus with simulated MG resulted in an increase in tube formation and angiogenesis. Our findings indicate that simulated MG driven angiogenesis was mediated by iNOS-cGMP pathway. Further, we checked whether microgravity sensitization maintained the functionality of endothelial cells. Towards this, we performed the tube formation assay of microgravity-treated endothelial cells. Microgravity-stimulated endothelial cells have shown to form more tubes as compared to cells under gravity. Next, we explored possibilities for the applications. We hypothesized that microgravity sensitized endothelial cells may prove to hasten wound healing process by re-establishing rapid angiogenesis at wound site. We examined the effect of microgravity on wound healing using rat model and observed that microgravity hastens the wound healing process as second degree burn wounds implanted with microgravity sensitized endothelial cells have shown to heal faster in rat model. Finally, we performed deep sequencing of miRNA followed by genome wide transcriptome sequencing of microgravity sensitized endothelial cells to unravel the mechanism of microgravity induced angiogenesis. An extensive analysis of the data including differential miRNA expression profile, heatmap showing relative expression of miRNA, comparative expression profile between miRNA and their mRNA targets detected under microgravity condition, were performed. The analysis documented up-regulation of miRNAs related to angiogenesis and their related pathways. Present study infers that microgravity sensitized endothelial cells promotes angiogenesis, which can be used as a primer to ensure an efficient wound healing process. An in-depth deep sequencing study confirms that microgravity sensitizes the genome of human endothelial cells to promote angiogenesis and thereby wound healing

Comparison of the modulation of FGFR signalling by thalidomide and its analogs lenalidomide and pomalidomide

Thalidomide, a powerful teratogen, re-emerged as a wonder drug for its teratogenic, anti-angiogenic and anti-tumor properties. Being FDA approved for Multiple Myeloma along with the analogs lenalidomide and pomalidomide is currently being tested in more than 2000 clinical trials for a range of conditions including solid tumors and inflammatory disorders. Fibroblast growth factor receptors (FGFRs) play key roles in embryonic development and cancer. There are indications that thalidomide might be linked to FGFR biology, however no experimental evidence is available till now. To understand the effects of thalidomide and its analogs, lenalidomide and pomalidomide, we utilized in silico predictive tools, kinome profiling, transcriptome and phophoproteome tools to study the modulation of FGFR signalling in endothelium. Genecodis and Enrichr with the differentially expressed genes were used to obtain the Gene Ontology, Transcription factor, Pathway and miRNA enrichments. The association of the drug with FGFR signalling was investigated at various levels. Protein-chemical network tool, STITCH and Pocketome predicted strong association of thalidomide with FGFR2. At gene expression level, FGFR1 and FGFR2 were found to be affected under the three drug treatments in in vitro and in vivo models. Kinomescan results suggest the binding of thalidomide with high affinity to a mutant FGFR3 (G697C) and FGFR2. To validate this, we checked the activity of FGFR2 kinase under the three drug treatments and found that they affected the kinase activity in a dose-dependent manner with pomalidomide having lowest IC50 value. Blind docking using Autodock revealed the possible binding sites and interestingly all the three analogs were predicted to bind to Lys517 of FGFR2. Lys517 is one of the ATP binding sites, suggesting that possibly analogs interfere with the ATP binding. Taken together, FGFRs could be potential targets of thalidomide and its analogs and the modulation of FGFRs by thalidomide partially explain the teratogenic and anti-tumor properties of the drug. Thus through different platforms, the mechanisms of drugs could be understood in a better way. This in turn will aid in modifying the drug structures resulting in the development of new analogs with more efficacy and reduced undesired effects

Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma

Inhibition of Angiogenesis and Nitric Oxide Synthase (NOS), by Embelin & Vilangin Using in vitro, in vivo & in Silico Studies

Purpose: In recent year’s anti-angiogenesis agents have been recognized as effective drugs for the treatment of solid tumors, this prompted us to conduct the present study. Methods: The anti-angiogenic activity of dimeric form of embelin (vilangin) was evaluated using endothelial cell (in vitro) and chorioallantoic membrane (CAM) egg yolk angiogenesis model (in vivo) and in addition the docking behaviour of human nitric oxide synthases (NOS) with four different ligands was evaluated along with their putative binding sites using Discovery Studio Version 3.1 (in silico) compared with the parent compound (embelin). Results: Vilangin exhibits 50% cytotoxic at 92 ± 1 µg/ml concentration level with reference to ECV 304 endothelial cells. Both vilangin and embelin, showed inhibitory effects on wound healing, single cell migration, nitric oxide production, and endothelial ring formation at 0.1 and 1.0 μg/ml concentration level. Similarly, CAM assay also showed inhibitory effect of vilangin and embelin with respect their reduction in length, size and junctions of blood capillaries compared to untreated egg yolk. Docking studies and binding free energy calculations revealed that vilangin has maximum interaction energy (-74.6 kcal/mol) as compared to the other investigated ligands. Conclusion: The results suggest that both vilangin and embelin attenuates angiogenesis in similar manner

Sinapic acid prevents hypertension and cardiovascular remodeling in pharmacological model of nitric oxide inhibited rats.

Hypertensive heart disease is a constellation of abnormalities that includes cardiac fibrosis in response to elevated blood pressure, systolic and diastolic dysfunction. The present study was undertaken to examine the effect of sinapic acid on high blood pressure and cardiovascular remodeling.An experimental hypertensive animal model was induced by L-NAME intake on rats. Sinapic acid (SA) was orally administered at a dose of 10, 20 and 40 mg/kg body weight (b.w.). Blood pressure was measured by tail cuff plethysmography system. Cardiac and vascular function was evaluated by Langendorff isolated heart system and organ bath studies, respectively. Fibrotic remodeling of heart and aorta was assessed by histopathologic analyses. Oxidative stress was measured by biochemical assays. mRNA and protein expressions were assessed by RT-qPCR and western blot, respectively. In order to confirm the protective role of SA on endothelial cells through its antioxidant property, we have utilized the in vitro model of H2O2-induced oxidative stress in EA.hy926 endothelial cells.Rats with hypertension showed elevated blood pressure, declined myocardial performance associated with myocardial hypertrophy and fibrosis, diminished vascular response, nitric oxide (NO) metabolites level, elevated markers of oxidative stress (TBARS, LOOH), ACE activity, depleted antioxidant system (SOD, CAT, GPx, reduced GSH), aberrant expression of TGF-β, β-MHC, eNOS mRNAs and eNOS protein. Remarkably, SA attenuated high blood pressure, myocardial, vascular dysfunction, cardiac fibrosis, oxidative stress and ACE activity. Level of NO metabolites, antioxidant system, and altered gene expression were also repaired by SA treatment. Results of in vitro study showed that, SA protects endothelial cells from oxidative stress and enhance the production of NO in a concentration dependent manner.Taken together, these results suggest that SA may have beneficial role in the treatment of hypertensive heart disease by attenuating fibrosis and oxidative stress through its antioxidant potential