Tipping off endothelial tubes: nitric oxide drives tip cells.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

Sinapic acid protects heart against ischemia/reperfusion injury and H9c2 cardiomyoblast cells against oxidative stress.

The present study was designed to evaluate antioxidant and cardioprotective potential of sinapic acid (SA) against ischemia/reperfusion (I/R) injury. Cardiac functional recovery after I/R was evaluated by percentage rate pressure product (%RPP) and percentage coronary flow (%CF). Myocardial injury was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining and LDH enzyme leakage. Oxidative stress was estimated by lipid peroxidation level. eNOS protein expression in reperfused heart was assessed using Western blot method. Finally, in order to support the antioxidant effect of SA, in vitro protective potential of SA was assessed on H2O2-induced oxidative stress in H9c2 cardiomyoblast cells. The overall results demonstrated that I/R induced cardiac dysfunction, injury and oxidative stress was attenuated by SA treatment. Moreover, in vitro results also shown that, SA protects H9c2 cells from oxidative stress and modulates mitochondrial membrane permeability transition (MPT). In conclusion, coupled results from both in vivo and in vitro experiments have confirmed that SA with antioxidant role protects cardiac cells and its functions from I/R induced oxidative stress.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium.

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO-sGC-cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients.