Studies On The Discovery Of New Drugs For Preventing And Treating Vascular Diseases

Cardio-cerebral-vascular diseases are threatening the health of human.About 20～30%people in the world are suffering for these diseases.The root of the different cardio-cerebral-vascular diseases is the pathological changes of the vessels,most of which can be classified into vascular diseases.Vascular diseases are the integrated outcomes for many different kinds of pathogenesis.Now,there are several different kinds of drugs for treating these vascular diseases,some of which have very good curative effects.However,most drugs need life-time administration and still can not change the mortality rate.The discovery of novel drugs for preventing and treating vascular diseases is the purpose of the work for all researchers worldwide.The purpose of our study was to evaluate the drug targets we were interested in, then establish a drug screening model,and finally find the active new lead compounds. Our research would be presented as the followings.Partâ… :Recombinant Expression of Chymase and High-throughput Screening for Its Inhibitors.Renin-angiotensin system plays an important role in pathology and development of many different vascular diseases.Besides angiotensin converting enzyme(ACE), chymase is another enzyme which can produce Angâ…¡from Angâ… .Recently,many studies propose that chymase might be a novel drug target for preventing and treating vascular diseases.Hamster heart chymase 2 belongs toαchymase group,just like human chymase. Hamster chymase 2 was revealed to be very similar in sequence of human chymase,thus hamster chymase 2 can be used as a substitute for human chymase.In order to further inspect chymase and develop its inhibitors,we firstly cloned and expressed hamster chymase 2 in Pichia yeast and E.coli.The protein expression level of chymase 2 is very low in Pichia yeast.However,the expression level was extremely high in E.coli.We also found the chymase 2 protein obtained from the E.coli had well activity of converting Angâ… to Angâ…¡.Then,we successfully set up a stable,sensitive,reproducible high-throughput screening model for searching its inhibitors.A total of 40080 samples were screened,of which 807 samples were screened again.We finally found 52 compounds and 102 Traditional Chinese Medicines to be chymase inhibitors,and there were two compounds with IC₅₀ less than 1μM.Partâ…¡:Evaluation of Urotensinâ…¡'s Vascular Activity and Establishment of A Mammalian Cell Line with Urotensinâ…¡Receptor Expressed StablyUrotensinâ…¡is the most potent vasoconstrictor found so far,and its receptor namely Urotensinâ…¡receptor was recently discovered,which was a G protein coupled receptor. Many studies have illustrated that Urotensinâ…¡possesses wide activity in cardiovascular system.Its receptor antagonists may be used to cure cardiovascular diseases such as hypertension and atherosclerosis in the future.Our study was to evaluate this new target to some extent.We found the desensitization to Urotensinâ…¡in rat thoracic rings.The second contraction induced by Urotensinâ…¡was significantly reduced,comparing to the first contraction.We observed that desensitization to Urotensinâ…¡was not significantly affected by endothelium denudation,however,it was significantly prevented by the NOS inhibitor L-NAME,demonstrating that the desensitization was related to the NOS activity in vascular smooth muscle.Subsequently,we discovered that iNOS inhibitor aminoguanidine could notably inhibit the desensitization to Urotensinâ…¡in endothelium-denuded aortic rings,but nNOS inhibitor spermidine could not.Finally,we suggested that the mechanism of this desensitization phenomenon should be that Urotensinâ…¡could activate iNOS in vascular smooth muscle,promoting the release of NO.We studied the effects of Urotensinâ…¡on the expression of several harmful vascular factors in rat vascular smooth muscle cell.We found that Urotensinâ…¡could up-regulate the mRNA expression of iNOS and VEGF,and the effect on VEGF expression was also related to free radical generation.There is emerging evidence that adventitial fibroblasts play a vital role in vascular remodeling.We also discovered that Urotensinâ…¡could induce vascular adventitial fibroblast phenotypic differentiation into myofibroblasts,αsmooth muscle actin was an important marker of myofibroblast.We observed that the expression ofα-smooth muscle actin was increased after 48 h treatment of Urotensinâ…¡(100μM),which was significantly inhibited by free radical scavenger.Our study suggested that Urotensinâ…¡may participant the progress of adventitial remodeling.In order to better study the function of Urotensinâ…¡receptor,we expressed the receptor in mammalian cells.Firstly,we cloned the Urotensinâ…¡receptor gene from human genomic DNA,and pcDNA3.1(+) was selected to construct the recombinant plasmid.Subsequently,we transfected the plasmid into CHO cells.After screening by G418,we finally successfully set up the mammalian cell line with Urotensinâ…¡receptor expressed stably,which would help to the realization of Urotensinâ…¡receptor's fuction and the discovery of binding compounds in the future.Partâ…¢:Discovery of A Vascular Active Compound from the Herb GL.GL is a Chinese herb which is used as a folk medicine for the treatment of hypertension.Previous study found that GL extract had antihypertensive activity in both SHR and normal rats.However,we unexpectedly found the GL extract had very potent vasoconstrictive effect on rat thoracic rings.To demonstrate the reason,taking the technology such as extraction,separation,vascular activity analysis and chemical structure identification together,we finally isolated and identified a new vascular active compound from GL.Partâ…£:Vasorelaxant Effects of Pinocembrin and the Mechanisms Involved.Natural product is one of the large origins of drug discovery.After screening some compounds,we found pinocembin had vasorelaxant activity.Pinocembrin(5～100μM) induced relaxation in aortic rings pre-contracted with NE or KCl.Pretreatment with pinocembrin(30 or 50μM) also inhibited contractile responses to NE and KCl.The vasorelaxant effect of pinocembrin relied on intact endothelium partially,and incubation with NOS inhibitor L-NAME or guanylate cyclase inhibitor methylene blue significantly inhibited the effect,however the cyclooxygenase inhibitor indomethacin had no influence on the action.In endothelium-denuded rings,the vasorelaxant effect of pinocembrin was reduced by K⁺ channel blockers glibenclamide,tetraethylammonium and 4-aminopyridine.Pinocembrin also reduced NE-induced transient contraction in Ca²⁺-free solution and inhibited contraction induced by increasing external calcium in Ca²⁺-free medium plus 60 mM KCl.Our results suggested that pinocembrin induced relaxation in rat aortic rings through an endothelium-dependent pathway involving NO-cGMP,and also through an endothelium-independent pathway by opening K⁺ channels and blockade of Ca²⁺ channels.In summary,the present study focused on the key steps of new drugs discovery such as drug target validation,high-throughput screening and hits' activity evaluation.Our study would promote the new drugs discovery for preventing and treating vascular diseases.