| BACKGROUND AND OBJECTIVEIschemic diseases, a series of diseases caused by insufficient artery blood supply mainly include peripheral arterial occlusive disease (PAOD), ischemic heart disease, and ischemic cerebrovascular disease. PAOD includes atherosclerosis obliterans, thromboangitis obliterans, arterial thrombosis and arterial embolism. PAOD is commen in vascular surgery. It is characterized by high disease, disability and death rates. In ischemic diseases, ischemia is more dangerous in the heart and brain. In many patients with ischemic diseases, revascularization by endovascular means or by open surgery, combined with best possible risk factor modification, does not salvage limbs or viscera or relieve ischemic rest pain. As a consequence, novel therapeutic strategies developed over the last two decades have aimed to promote neovascularization and remodelling of collaterals. In other words, therapeutic angiogenesis is important to these patients with ischemia. Indeed, stimulating angiogenesis could help reduce the damage to ischemic tissues. Many ischemic diseases such as ischemic coronary artery disease, critical limb ischemia and brain infarction may benefit from the induction of angiogenesis. However, aberrant or excessive angiogenesis allows for vascularization of solid tumors and provides routes for metatasis of cancer cells. A better understanding of the steps controlling angiogenesis under the ischemic state should further advance our attempts to stimulate or inhibit angiogenesis when warranted.The use of small cell-permeable molecules to effect biological phenomena, also known as chemical genetics, has made a significant impact in diverse areas of biological medicine. Our recent studies have used chemical biology to discover novel small-molecule angiogenic promoters. These chemical molecules could be used as tools to clarify the mechanism of angiogenesis. Furthermore, they can be used as potential drugs for therapeutic angiogenesis. Vascular endothelial cells (VECs) are an attractive target for therapeutic angiogenesis because they are intimately involved in disease processes associated with angiogenesis. Initiation of angiogenesis involves migration and proliferation of VECs. Finding small molecules that could promote VEC migration and induce angiogenesis is crucial for patients and for researchers.When deprived of serum and FGF-2, in vitro-grown HUVECs gradually detach from the Petri dish and undergo apoptosis. This in vitro model is often used to mimic the in vivo ischemic state and has been widely used. Our previous study showed that a novel pyrazole derivative, ethyl3-(o-chlorophenyl)-5-methyl-l-phenyl-lH-pyrazole-4-carboxylate (MPD) at 20 and 25μM increased VEC viability and inhibited VEC apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2). Moreover, MPD at 5 and 10μM did not affect VEC viability or apoptosis induced by deprivation of serum and FGF-2. In this study, we used this model to investigate whether MPD could promote angiogenesis under the ischemic state at these four concentrations.In vivo, NO is produced by catalysis of 1-arginine by endothelial nitric oxide synthase (NOS), a calcium-dependent enzyme involved in angiogenesis. NO presumably acts as a vasodilator to result in arteriolar genesis (formation of muscular arterioles) and angiogenesis (formation of new blood vessels, mainly capillaries). Diminished bioavailability of NO is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis, vasoconstriction, hypertension, atherosclerosis, and PAOD. In vitro, NO promotes most angiogenesis-related properties of endothelial cells, including migration and growth, as well as formation of capillary-like structures. Increased NO bioavailability can enhance endothelial cell survival, thus facilitating ischemia-mediated angiogenesis. NO plays a central role in angiogenesis. Therefore, we use MPD to investigate the roles of NO in angiogenesis.ROS are ubiquitous, highly diffusible and reactive molecules produced by the reduction of molecular oxygen. They are normally produced during the respiratory burst of phagocytes as a defense mechanism against pathogens. More recently, vascular cells have been found to produce ROS, and these molecules are implicated in endothelial dysfunction associated with atherosclerosis and ischemia-reperfusion injury. ROS participates in the apoptosis of VECs and have been suggested as important mediators for angiogenesis. ROS stimulates angiogenesis in vitro. In vivo, elevated oxidative stress is directly associated with neovascularization and vascular endothelial growth factor (VEGF) expression in aortic plaque of models of atherosclerosis. ROS play an important role in angiogenesis both in vitro and in vivo. They are produced in response to hypoxia, ischemia, and angiogenic growth factors such as VEGF and angiopoietin-1, thereby stimulating VEC proliferation and migration. However, whether MPD modulates angiogenesis through ROS is unknown.Interferon-inducible protein 10 (CXCL10/IP-10) is a CXC chemokine produced by certain types of cells, including activated endothelial cells, monocytes, fibroblasts, and keratinocytes. IP-10 is a potent inhibitor of angiogenesis. IP-10 inhibits proliferation of endothelial cells in vitro. IP-10 inhibits endothelial cell growth and is inversely correlated with VEGF production. IP-10, added to HUVECs, cultured on a Matrigel substrate, inhibits their differentiation into tube-like structures in a dose-dependent fashion and reduces the extent of the neo-vascular network; IP-10 neovascularization in animal models. In summary, IP-10 plays an import role in angiogenesis both in vitro and in vivo. It has been proposed that this chemokine is also involved in the pathogenesis of cardiovascular diseases and coronary syndromes. There is growing evidence implicating this chemokine in both infectious and noninfectious causes of injury. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. However, whether IP-10 is associated with ischemic state and modulates angiogenesis is not clear.The utilization of chemical genetics may discover novel key factors involved in angiogenesis. Therefore, it can provide experimental evidences to illustrate issues mentioned above. However, the molecular mechanisms of VEC angiogensis remain unclear. They need further study. Based on the backgrounds mentioned above, the objective of this study was as follows:To investigate the roles of NO, ROS and IP-10 in VEC angiogenesis by using MPD, and the molecular mechanisms mediated by these three elements. The researches would provide a theoretical basis for studying the molecular mechanisms of VEC angiogenesis. Our study provides potential drug for therapeutic angiogenesis of ischemic diseases. Moreover, our data would provide provided new clues and targets for ischemic diseases therapy in clinic.STUDY CONTENTS1. Study of whether MPD could inhibit VEC apoptosis in vitro ischemic model (HUVECs cultured without FGF-2 and serum).2. Study of whether MPD could promote angiogenesis in vitro ischemic model3. Study of the molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPDMETHODS:1. Vascular endothelial cell culture:HUVECs were obtained as described before by [Jaffe et al.,1973].2. Cell apoptosis analysis:1) observation of cell morphological changes by phase contrast microscope2) cell viability was determined by MTT-assay3) analysis of nuclear fragmentation and chromatin condensation by the acridine orange staining combined with (laser scan) confocol microscope3. Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously [Kureishi Y et al,2000]4. Cell migration assay in vitro:monolayer cell wound healing assay as described previously [Burk,1973; Vasvari et al,2007]5. NO production assay:NO detection kit6. Analysis ROS level by the fluorescence probe (DCHF) combined with laser scan confocol microscope.7. Analysis of expression and distribution of proteins:1) examination of the changes in Alix protein level and distribution by immunocytochemistry combined with laser scan confocol microscope2)Analysis the levels of Alix, Ets-1 and IP-10 by Western blot assay.RESULTS:1. MPD inhibited VEC apoptosis induced by deprivation of serum and FGF-2. Morphological changes of VECs were observed with phasecontrast microscopy. MPD at 20 and 25μM could inhibit VEC apoptosis induced by deprivation of serum and FGF-2 at 24 h. On the contrast, MPD at 5 and 10μM did not affect VEC viability and apoptosis induced by deprivation of serum and FGF-2 at 24 h., The number of apoptotic bodies decreased obviously with treatment of MPD (25μM) under phase microscope. AO staining showed that MPD (25μM) inhibited nuclear fragmentation and chromatin condensation.2. MPD promoted angiogenesis in vitroischemic model.2.1 To demonstrate the angiogenic-inducing function of MPD in HUVECs, we assayed capillary-like tube formation on Matrigel. Capillary-like tubes developed well in HUVECs treated with MPD (5 and 10μM) on Matrigel-coated 24-well plates with basal M199 medium at 6,12 and 24 h. HUVECs treated with MPD at 5 and 10μM could form tubes (* p<0.05 or** p<0.01). However, HUVECs treated with MPD at 20 and 25μM could not form tubes well.2.2 We performed monolayer cell-wound healing assay to determine whether MPD affects HUVEC migration. MPD at 5 and 10μM promoted cell migration(*p<0.05 or** p<0.01), with the gap nearly closed at 24 h. MPD at 20 and 25μM could not promote cell migration obviously.3. The molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPD3.1 We determined the level of NO in MPD-induced angiogenesis after HUVECs were treated with MPD for 6,12 and 24 h. NO production was moderately elevated in HUVECs treated with 5 and 10μM (* p<0.05) MPD and extremely elevated with 25μM MPD (** p<0.01) at 6 and 12 h. MPD at 5,10 and 25μM did not affect the level of NO at 24 h(p>0.05). 3.2 We detected the levels of intracellular ROS in HUVECs treated for 6,12 and 24 h. The level of ROS was increased in cells treated with 5 and 10μM MPD (* p <0.05) but was decreased in cells treated with 25μM MPD (* p<0.05) as compared with controls.3.3 We investigated the effects of MPD on IP-10 in VECs. When VECs were treated with 5,10 and 25μM MPD for 24 h, the level of IP-10 was much lower than that of the control group (p<0.05); MPD at 5,10 and 25μM did not affect the level of IP-10 at 6 and 12 h compared to the control group (p>0.05)CONCLUSIONS:1. MPD could both inhibit VEC apoptosis and promote VEC angiogenesis in vitro ischemic model.2. In vitro ischemic model, MPD at 5 and 10μM promoted VEC migration and angiogenesis by upregulating NO and ROS levels and depressing IP-10 level. MPD at 25μM inhibited VEC apoptosis by greatly upregulating NO level and depressing ROS and IP-10 levels.3. ROS had close relationship with NO and both of them were associated with apoptosis and angiogenesis. MPD at different concentrations probably effected related factors involved in this signal transduction pathway, that is, controlling ROS and NO levels, to inhibit VEC apoptosis induced by deprived of serum and FGF-2 and promote angiogenesis.4. MPD could inhibit the expression of IP-10. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. Therefore, our data indicated that MPD could reduce damage in ischemic state by depressing the level of IP-10. IP-10 may become a new target for future clinical treatment of ischemic diseases.5. MPD is a good tool for investigating the difference of cell differentiation and apoptosis and for investigating the mechanisms of VEC apoptosis and angiogenesis, and MPD might be useful in the development of new drugs in therapy of ischemic diseases. Moreover, it also provides new clues and target for clinical treatment of ischemic diseases. |
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