| ObjectionDrug, ,PCI and CABG are the main therapeutic approaches of ischemic cardiac disease. However, they are less effective to ischemic cardiac disease in late phase. Furthermore, restenosis occurs in the 30 - 50% target vessels after operation. PCI and CABG are not suitable for some patients because coronary artery lesions are diffuse or sites of obstruction lie on small blood vessels. How to treat the patients of ischemic cardiac disease unsuitable to operation and ischemic cardiac disease in late phase become the very hot topic in nowadays medicine field. Therapeutic angiogenesis means to stimulate the fonnation of new capillaries net works connecting with the original capillaries at ischemic area by drugs, gene therapy or others. The angiogenesis improve the ischemic situation by means of promoting and (or) increasing the growth of collateral arteries. This kind of method becomes one of the hot topics in nowadays, including many basic and clinic studies. The vascular growth effects of various angiogenetic factors and their transgenic factors have been confirmed. However their application are limited because of the limitation of vascular growth factors ,their complicated application and lots of problems existing in transgenic therapy. So we are searching for the more convenient, effective and useful drugs to induce angiogenesis for substituting the growth factors. Some specialists focus on the non - lipid - regulatory effects of statins.Statins are 3 - hydro 3 - methylglutaryl coenzyme A ( HMG - GoA) reduc-tase inhibitor developed in recent ten years, whose development improves the lipid - lowering drug therapy greatly. The recent clinic experiments and basic studies reveal that statins can not only lower the levels of serum cholesterol and low density lipoprotein cholesterol, but also block atherosclerotic process, and lower the mortality rate of the CHD (coronary heart disease) patients, and de-crease the re - operation rate after PCI and occurrence of cerebral stroke. These benefits come from non - lipid - regulatory effects independent of statins lipid -lowering characteristics. Recently, it is found that many kinds of statins have non - lipid regulatory angiogenetic effects independent of their lipid - lowering characteristics. Statins are taken as the remedial angiogenetic drug. They may bring the new therapeutic approaches for the patients of late phase ischemic cardiac disease intolerant to operation. Therefore, this approach receives much attention from researchers all over the world. Pitavastatin, synthesized by Japanese chemical industry and developed clinically by Yuhe Family Company, is one of new powerful cholesterol - lowering lipophilic statins. Its construction is similar to Atorvastatin. This paper discusses the angiogenetic effect and mechanism of Pitavastatin in vivo or vitro, and provides theoretic basis of statins as the therapeutic angiogenesis drugs in clinical application.Methods1. Experiments in vivo1.1 Animal models and grouping1.1.1 45 male wild - type C3H/He mice weighting 22 - 33 g,aging 32 -48 weeks are randomly divided into 3 groups: (1) Control group, (2) Pitavastatin group(1mg/kg/d) , (3) Atorvastatin group(10mg/kg/d).8 male Klotho gene - knockout mice (Homo kl - / - ) weighting 6 - 12g, aging 5 weeks, are randomly divided into 2 groups: (1) Control group, ( 2 ) Pitavastatin group (1mg/kg/d).24 male Klotho gene - knockout partially mice ( hetero kl + / - ) weighting 22 -33g, aging 10-12 weeks, are randomly divided into 4 groups: (1) hetero (kl +/ - ) control group, (2) hetero (kl +/ - ) treating group (lmg/kg/d Pitavastatin) , (3) wild (kl+/ + ) control group, (4) wild (kl+/ + ) treating group (1mg/kg/d Pitavastatin).1.1.2 Ischemic hind - limb models of mice: The distal portions near knees and proximal portions of femoral arteries and veins in C3H/He and Klotho (kl+/-, kl+/ + ) mice are ligated, exfoliated and cut off after the mice havebeen treated by statins for a week. Then all mice are treated continuously for next 5 weeks.1.2 Measurement of indexes and collection of samples 1.2. 1 Monitoring of hind - limb blood flow: The blood flow of bilateral hind - limbs of C3H/He and Klotho (kl+/-, kl+/ + ) mice are measured by means of Laser Doppler perfusion imager ( LDPI) respectively 1 week before ischemic operation of hind - limbs ( - 1 week), the time of operation (0 week) , and 1,2,3 ,4 and 5 week after operation (1,2,3,4 and 5 week). The ratio of blood flow of ischemic (right) limb to normal (left) limb (Rt/Lt) is calculated by means of Moor LDI Image Processing V3. 01.1. 2. 2 Measurement of hemo - biochemical indexes: In the 5th weeks after ischemic operation of hind - limbs, serum separated from 1ml blood of post -orbital venous plexus is stored in the fridge at -80℃. Serum lipids,hepatic and renal functions , creatine phosphokinase (CPK) and nitric oxide metabolite NOx ( NO3- + NO2- ) are determined by Hitachi 7071 type auto - analysis apparatus.1. 2. 3 Pathological samples: After the blood of post - orbital venous plexus is collected in the 5th weeks after operation, the mice are killed. Their muscles cut partially from the proximal portions of thigh are stored in deep - frozen fridge at - 80℃, while the expression of vascular endothelial growth factors (VEGFs) proteins in ischemic limbs are observed by the way of Western blotting. Meanwhile, other muscles cut from the proximal portions of thigh are fixed in methanol, dehydrated gradually, embedded in paraffin and made into serial section. All sections are prepared for pathological morphologic observation and immuno - histochemical analysis (immunofluorescence and immuno - enzyme histochemical technique). Immunofluorescence histochemical technique applied to SP method. The numbers of capillaries is measured by systemic biomicro-scope. Immuno - enzyme histochemical technique used direct method. The number of phospho - Akt positive cells is measured by systemic biomicroscope. The fat in liver and spermary are separated and weighted.2. Experiments in vitro2.1 Co - culture of neo - angiogenesis2.1.1 Many kinds of statins culture: Human umbilical venous endothelial cells (HUVEC) are co - cultured with fibroblasts separately added 10%DMSO (control group),10-8 10-10mol/ml Pitavastatin,10-6 10-8mol/ml Atorvastatin,10-5 10-7 mol/ml Pravastatin (positive groups) or 25μl Suramin (neo- angiogenesis inhibitor) into multiple 6 holes Petri dish. The numbers of vessel- like structures in each hole are calculated and compared by TMS.2.1.2 Pitavastatin culture: Its concentration is from 10-10 to10-13mol/ ml. We calculate and compare the Area, Length, Joints and Path of vessel -like structures by neo - angiogenesis quantitative analysis software system (Ver1.0).2.2 Culture of Human umbilical venous endothelial cells ( HUVEC ) HUVECs are inoculated in two 6 - cells culture plate with the density of 5 10 cells per hole, divided into: ①control group; ②10-13mol/ml Pitavastatin; ③10-12mol/ml Pitavastatin; ④10-11mol/ml Pitavastatin; ⑤10-10mol/ml Pitavastatin; ⑥10-9 mol/ml Pitavastatin; ⑦ 10-8 mol/ml Pitavastatin; ⑧ 100ng/ml VEGF (positive control group). HUVEC are retrieved after culture. The expressions of VEGF,bFGF,p - Akt and eNOS proteins are observed by Western blotting. Meanwhile culture medium is retrieved, and the content of NOx in medium is measured by cadmium reduction Griess method.Statistical analysisAll of the date are expressed by means ± standard deviation ((x|-) ± s). The means of samples (among groups or internal groups) are compared by t test. And P<0.05 is considered as significant difference.Results1. Experiments in vivo1.1 The effects of statins on the levels of serum lipids, hepatic and renal function, CPK of C3H/He and Klotho mice: There are no obvious effects of statins (Pitavastatin, Atorvastatin) on these indexes, In the 5th weeks after oper-ation.1.2 The effects of Pitavastatin on the contents of NOx in the serum of C3H/He mice: Pitavastatin made the contents of NOx in the serum of C3H/He mice are significantly higher than those of control group.1.3 The effects of statins on the reperfusion in the ischemic limbs of C3H/He and Klotho miceThe results of blood flow of C3H/He and Klotho mice in each group before or after operation by means of Laser Doppler perfusion imager (LDPI) :1.3.1 The effects of statins on the reperfusion in the ischemic limbs of C3H/He mice: In statins groups especially Pitavastain group, the blood flow of the ischemic limbs recovered gradually one week after operation, and approached to the levels of pre - operation 3-4 weeks after operation. The ratio of blood flow of ischemic (right) limb to normal (left) limb (Rt/Lt) is obviously increased.1.3.2 The effects of statins on the reperfusion in the ischemic limbs of Klotho mice: After treated by Pitavastatin, the blood flow of ischemic limbs in both Klotho(Kl +/ + )and Klotho(Kl +/ - )groups recovered obviously.1.4 The effects of Pitavastatin on the density of capillaries in ischemic limbsThe results of immuno - histochemical method (immunofluorescence histo-chemical technique, SP method) : Pitavastatin could increase the density of capillaries in ischemic limbs of C3H/He and Klotho mice.1. 5 The effect of Pitavastatin on the activity of protein kinase B ( PKB, also called Akt) phosphatase in ischemic limbs:The results of immuno - histochemical method (immuno - enzyme histochemical technique, direct method) : Pitavastatin could increase the activity of Akt in ischemic limbs of C3H/He and Klotho mice (increased the number of p - Akt positive cells obviously)1. 6 The effects of Pitavastatin on the expression of VEGF protein of C3H/He and Klotho mice:The results of Western blotting revealed that Pitavastatin could enhance the expression of VEGF protein of ischemic limbs of C3H/He and Klotho mice.1. 7 The effects of statins on the body weight and weight of visceral organs in C3H/He and Klotho mice:Statins ( Pitavastatin, Atorvastatin) had no obvious effects on the body weight and weight of visceral organs in C3H/He mice.Pitavastatin had no obvious effects on the body weight and weight of visceral organs in Kl(+/-,+/ + ) groups before or after experiment.2 Experiments in vitro2.1 Co - culture of neo - angiogenesis2.1.1 Statins culture: with 10-10mol Pitavastatin, 10-6mol Atorvastatin, 10-5mol Pravastatin, vessel - like structures grew well, and their numbers of the lumen is significantly higher than that of both control and neo - angiogenesis inhibitor groups. But it had no significant difference from positive control (VEGF) group.2.1.2 Pitavastatin culture: With the Pitavastatin concentration of 10-11 10-10mol, Area of the lumen of vessel - like structures is significantly higher than control group, but of no significant difference from positive control (VEGF) group. With the Pitavastatin concentration of 10-10mol, Area, Length, Joint and Path of the lumen of vessel - like structures increased significantly. But it has no significant difference from positive control (VEGF) group.2. 2 Culture of Human umbilical venous endothelial cells (HUVEC) 2. 2.1 The effects of Pitavastatin on the content of NOx in HUVEC medium : With the Pitavastatin concentration of 10-9 10-12mol, the content of NOx in HUVEC medium enhanced obviously, but of no significant difference from positive control (VEGF) group.2.2.2 The effects of Pitavastatin on the expressions of VEGF,bFGF,p -Akt and eNOS proteins: The results of Western blotting revealed that with the Pitavastatin concentration of 10-10 10-12mol, the expressions of VEGF,bFGF, p - Akt and eNOS proteins enhanced.DiscussionIt is approved that statins had the clear - cut effects to regulate the disorderof serum lipids and lower the CHD events. Furthermore, statins effects on several aspects, to improve the prevention and cure of atherosclerosis dramatically. However, the anti - atherosclerosis effects of statins did not accord with the lowered level of cholesterols. Except the effects of non - regulatory anti - atherosclerosis of statins, Laufs U found that obstructional site caused by the same occlusion vessels could contracted, after treated by statins, and it could be associated that statins could promote the formation of collateral circulation of ischemic tissue. Masataka Sata revealed that the collateral blood flow and capillaries number of muscles of ischemic limbs increased obviously, after treated by statins. It is found that statins had other effects to improve the blood flow and angiogenesis of ischemic tissue independent of its non - lipid regulatory characteristics. Whether this effect became the therapeutic angiogenesis method is paid wide attention by researchers in or abroad.After advanced aging C3H/He wild - type ischemic mice are treated by Pi-tavastatin, their collateral blood flow and the density of capillaries in ischemic limbs increased obviously, which revealed that Pitavastatin could accelerate the formation of collateral circulation and improve the angiogenesis of ischemic limbs. Pitavastatin might take part in the angiogenesis by means of two mechanisms , and first of all, one of them is promoting the secretion and release of angiogenesis factors. Recently, many laboratories have confirmed the angiogenesis effects of vascular growth factors and their trans - gene therapy in ischemic tissues. Among them, the most representative one is VEGF. VEGF is a kind of highly specific, powerful intravascular dividing promoting factor and angiogenesis factor, and it directly, specifically had effects on vascular endothelium and promoted the proliferation and migration of vascular endothelial cells and formation of collateral vessels. Then basic fibroblast growth factors (bFGF) could also promote the proliferation and migration of vascular endothelial cells and angiogenesis. Our experiment in vivo found that the expression of VEGF proteins of ischemic limbs in treating groups enhanced, which revealed that Pitavastatin produced a marked effect by the pathway of promoting the secretion and release of angiogenesis factors. Secondly, the increasing of the content of NO in the blood and the activity of p - Akt enzyme by immuno - enzyme histochemical a-nalysis in treating groups revealed that Pitavastatin could promote the production of NO by enhancing the activity of p - Akt by the pathway of enhancing the conduction of PI3K/Akt signals. Kureishi revealed that statins could enhance the activity of phospho - Akt, while Akt could promote the phosphotation of endo-thelial - type NOS. Masataka Sata revealed that statins took part in the improvement of blood flow in ischemic tissue by improving the increasing synthesis and release of NO leading to expanding of small vessels. Meanwhile, the blood flow of eNOS gene - knockout ischemic mice did not improve after treated by statins, which revealed that one of the mechanism of statins to improve the blood flow is the increasing of secretion and releasing of NO. Our experiment and some literatures had the similar result, which is Pitavastatin promoted the angiogenesis by means of PDK/Akt - NO pathway. Some reports revealed that VEGF promoted the production of NO dependent of PDK/Akt pathway, which coincided with our results of experiments, that is Pitavastatin could also affect by means of VEGF -PDK/Akt -NO pathway.Klotho(Kl) gene is senile inhibiting gene discovered by Matsumura in 1997 in the study of initial hypertension. The changes of biological phenotype of Klotho gene - knockout mice including: (1) blockage of growth and shortening of life; (2) reduce of activity and chaotic gait; (3) shrink of reproductive organs; (4) atherosclerosis and heterotopic calcification; (5) disorder of skeletal metabolism; (6) shrink of skin; (7) emphysema, and so on. With the increasing of the age, atherosclerosis became heavier gradually, while athrosclerosis consisted of the etiologic basis of ischemic cardiac heart disease. Fukino reported that the blood flow of ischemic limbs in Klotho gene - knockout partially (kl + / - ) mice recovered slowly, and the speed is significantly lower than that of wild - type ( kl + / + ) mice born in the same den. Pitavastatin had the obvious effect on the angiogenesis of advancing ageing wild - type ischemic mice, and what about its effect on the Klotho gene - knockout ischemic mice with severe atherosclerosis? Because of the short life of ( homo kl -/ - ) mice (with the mean age of 8 weeks) , they are treated by Pitavastatin for only 2 weeks. And their atherosclerosis improved, and capillaries number of ischemic limbs increased after treated for 2 weeks. The blood flow of ischemic limbs in Klotho( Kl+ / - ) groups recovered obviously, and their density of the capillaries in ische-mic limbs muscles obviously increased. The results above seconded only to the wild - type mice born in the same den, furthermore, the enhance of the expression of VEGF proteins in ischemic limbs and the activity of p - Akt revealed that Pitavastatin could also promote the angiogenesis of Klotho gene - knockout ischemic mice.The results of the co - culture of neo - angiogenesis revealed that not only Pitavastatin but also Atorvastatin and Pravastatin could promote the growth of the lumen of vessel - like structure, which illustrated that many kinds of statins could promote the growth of blood vessels. But their concentrations are different, for example, the effects of 10-10mol/ml Pitavastatin or 10-6 mol/ml Atorvastatin or 10-5mol/ml Pravastatin on the growth of the lumen of vessel - like structure are nearly the same. The Culture of Human umbilical venous endothelial cells ( HUVEC ) experiment revealed that with the stimulation of Pitavastatin, especially the concentration of 10-12,10-11mol/ml Pitavastatin, the enhancing of the expression of the VEGF and bFGF proteins revealed further that Pitavastatin might take part in the angiogenesis by promoting the secretion, release and production of VEGF and bFGF from the vascular endothelial cells. NO syn-thase (NOS) took part in the regulation of synthesis of NO as rate - limiting enzyme of NO. NO could diastole the blood vessels, regulate the tension of vessels , inhibit the adhesion and aggregation of platelets by the GMP - dependent and cGMP - independent pathway. Simvastatin and Pravastatin could enhance the expression of the mRNA of NOS in human saphenous venous endothelial cells, and cause the increasing of the NOS activity; Silivastatin could induce the expression of eNOS gene in endothelial cells and enhance the activity of NOS. In our experiment, in the 10-12 - 10-9 mol/ml Pitavastatin, the content of NO in the medium is significantly higher than that of control group, and the expression of p - eNOS protein enhanced, this effect is similar to the effects of other statins, which revealed that Pitavastatin effected by increasing the synthesis, secretion and release of NO. Further studies revealed that statins increased NO by stabling the mRNA of eNOS, inhibiting its secretion and increasing the expression of RNA. Our experiment revealed that after treated by Pitavastatin, espe-...
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