Studies Of Angiogenesis And Expression Of FIZZ1, VEGF And HIF-1α In Murine Models Of Asthma

The airway remodeling is a distinctive feature of asthma,characterized by hypertrophy and hyperplasia of airway smooth muscle,increase of mucous gland, thickening of the reticular basement membrane,and qualitative and quantitative changes of airway blood vessel.The increase in the number and size of vessel contributes to the thickening of airway wall,which in turn leads to critical narrowing of bronchial lumen.More recent studies found an increase in the total number of vessels in patients with asthma when compared with control subjects.Angiogenesis plays an important role in airway remodeling in asthma,while the mechanisms underlying this process are not fully elucidated.Some cytokines are involved in airway allergic inflammation,and also in the formation of vessels,such as found in inflammatory zone 1(FIZZ1),vascular endothelial growth factor(VEGF)and hypoxia-inducing factor 1α(HIF-1α).FIZZ1,a novel cysteine-rich secreted protein,is able to promote proliferation and migration of pulmonary endothelial cells,and is able to upregulate the expression of VEGF.VEGF is known to be one of the most important cytokines in the formation process of vessels,and it is the important down-stream cytokine of HIF-1a.HIF-1a is an essential transcription factor,whose expression and activity are tightly regulated by cellular oxygen concentration.In hypoxia,HIF-1a can increase VEGF expression and induce angiogenesis to satisfy the oxygen requirement.According to these reports, FIZZ1,VEGF and HIF-1αare supposed to play a key role in angiogenesis of asthma.Inhaled corticosteroid such as budesonide is the effective drug for asthma, alleviating airway allergic inflammation and reducing the thickness of the basement membrane.To date,there are some studies on the effects of inhaled corticosteroid in angiogenesis,while its mechanisms of the anti-angiogenic effect in asthma are to be elucidated.In this study,murine models are established by ovalbumin(OVA) sensitization and repetitive challenge.Then we are to investigate the changes of airway angiogenesis,the expression of FIZZ1 and VEGF in mouse lung tissues,and further the effects and possible mechanism of FIZZ1 in angiogenesis of asthma in PartⅠ.In PartⅡ,we are to establish murine models of chronic asthma,and to investigate the effects of budesonide on airway angiogenesis,and also the relationship between angiogenesis and HIF-1α,VEGF expression.PartⅠFound in inflammatory zone 1 influences angiogenesis in murine models of asthmaObjectiveAngiogenesis plays an important role in airway remodeling in asthma,while the mechanisms underlying this process are not fully elucidated.Found in inflammatory zone 1(FIZZ1),a novel cysteine-rich secreted protein,is able to promote proliferation and migration of pulmonary endothelial cells,and is able to upregulate the expression of vascular endothelial growth factor(VEGF),which is one of the most important cytokines in the formation of vessels.In this study,the role of FIZZ1 and VEGF in angiogenesis of asthma is to be investigated.MethodsMurine models of asthma were established by ovalbumin(OVA) sensitization on days 1 and 14 and OVA challenge beginning on day 21.Mice were divided into six groups as follows(n = 10):OVA-challenged mice for 7 days,14 days,and 28 days respectively,and healthy mice challenged with PBS for 7 days,14 days,and 28 days respectively.The expression of FIZZ1,VEGF,and von Willebrand factor (vWF)-stained vascular area were measured.Histologic examination was also performed on airway inflammation.Results1.There were obvious allergic inflammation in murine models of asthma,and the inflammation scores were increased significantly compared with those of healthy mice.2.vWF-stained vascular area was largely increased after 7-day OVA challenge in models of asthma when compared with that of healthy mice,reduced from day 14 of OVA challenge,and was reduced to the normal levels after 28-day challenge.3.Levels of FIZZ1 were increased significantly in models of asthma,reached the peak by 7-day OVA challenge,declined on day 14,and were reduced further after 28-day OVA challenge.4.VEGF expression was increased after 7-day OVA challenge in models of asthma when compared with that of healthy mice.And VEGF expression was reduced to the normal level after 14-day OVA challenge,and was reduced further after 28-day challenge.5.Vascular area was positively correlated with expression of both FIZZ1 and VEGF.And there was positive correlation between FIZZ1 expression and VEGF expression.ConclusionThere is obvious angiogenesis in lung tissues of murine models of asthma.FIZZ1 expression was increased,and was significantly correlated with vascular area and VEGF expression,suggesting that FIZZ1/ VEGF passageway plays a key role in angiogenesis of asthma. PartⅡEffects of budesonide on the expression of hypoxia inducible factor-1αand vascular endothelial growth factor in murine models of chronic asthmaObjectiveAirway inflammation in asthma is usually accompanied with formation of new blood vessels.Angiogenesis plays an important role in airway remodeling in asthma. Hypoxia-inducing factor 1α(HIF-1α) is an essential transcription factor in hypoxia, whose expression and activity are tightly regulated by cellular oxygen concentration. HIF-1αcan increase VEGF expression and induce angiogenesis to satisfy the oxygen requirement in hypoxia.VEGF is known to be one of the most important cytokines in the vessel formation.And inhaled corticosteroid,such as budesonide,is the effective drug for asthma,while the mechanisms of its effects on angiogenesis are not fully elucidated.In the present study,we applied inhaled budesonide to murine models of chronic asthma,and to elucidate the effect of budesonide on airway angiogenesis and on the expression of HIF-1αand VEGF.MethodsWe established murine models as follows:mice were sensitized on days 1 and 14 by ovalbumin(OVA) and challenged with OVA beginning on day 21.Mice were divided into three groups(n=10):models of asthma,budesonide-treated mice (100ug/kg.d).and healthy mice.The expression of HIF-1α,VEGF,and von Willebrand factor(vWF)-stained vascular area were measured using immunohistochemistry and Western blot,and airway histologic changes were also examined by hematoxylin & eosin staining and Masson Trichrome staining.Results1.In lung tissues of models of asthma,vascular area was increased,and was reduced significantly after administration of budesonide.2.HIF-1αexpression was elevated in models of asthma when compared with that of healthy mice,which was significantly reduced in budesonide-treated mice.3.VEGF expression was also elevated in models of asthma when compared with that of healthy mice,and was reduced in budesonide-treated mice.4.Vascular area was positively correlated with expression of both HIF-1αand VEGF.And there was positive correlation between HIF-1αexpression and VEGF expression.5.There were obvious allergic inflammation and collagen deposition in murine models of chronic asthma,and the inflammation scores and fibrosis scores were increased significantly,which were alleviated in budesonide-treated mice.ConclusionBudesonide inhibits angiogenesis,also the HIF-1αand VEGF expression in asthma. Vascular area was positively correlated with expression of both HIF-1αand VEGF.And there was positive correlation between HIF-1αexpression and VEGF expression.The results indicate that HIF-1α/VEGF passageway plays an important role in the anti-angiogenic effects of budesonide. PartⅢThe effects of conditioned immune response in murine models of chronic asthmaObjectiveConditioned immune response is also called the conditioned reflex immunoreaction, pairing a novel conditioned stimulus(CS) with unconditioned stimulus(UCS) that makes immune changes in organism.After CS-UCS pairings,re-exposure of animals to the CS alone results in conditioned immune response usually ascribed to the effects of UCS.Behavioral conditioning has been demonstrated to regulate both humoral and cellular immunity.However,its effects on asthma are not clear.In our experiment,we established murine models of chronic asthma,and applied tone to mice as CS,inhaled budesonide(BUD) and salbutamol(SAL) as UCS,and observed the effects of conditioned immune response in murine models of chronic asthma.MethodsChronic murine models were established as follows:mice were established by ovalbumin(OVA) sensitization on days 1 and 14 and OVA challenge beginning on day 21.Models of asthma were divided into groups as follows and received different treatments from day 28:In CS+UCS group(A),tone was applied as CS,inhaled budesonide and salbutamol as UCS,concitioned immune response models were established.In daily UCS group(B):mice were inhaled budesonide and salbutamol daily.In low-dose UCS group(C):mice were inhaled budesonide and salbutamol for the first 7days,then once a week.In CS group(D):mice were given tone daily.In OVA group(E):mice were the control models of chronic asthma.The mice in PBS group(F) were sensitized and challenged with PBS instead of OVA as the healthy controls. After 20-week different treatments beginning on day 28,the breath frequency of mice was recorded.Airway histologic changes were also examined by hematoxylin & eosin staining and Masson Trichrome staining.Results1.Breath frequency in the group A(CS+UCS group) and the group B(daily UCS group) was much reduced than that in the group E(OVA group).There was no significant difference between the group A and B.The breath frequency was much increased in the group C(low-dose UCS group),group D(CS group) and group E when compared with that in the group F(PBS group ). 2.Airway allergic inflammation in lung tissues was alleviated in the group A and group B when compared with those in the group E.The inflammation scores were much more in the group C,group D and group E when compared with those in the group F.3.Airway collagen deposition in lung tissues was alleviated in the group A and group B when compared with those in the group E.The fibrosis scores were much more in the group C,group D and group E when compared with those in the group F.ConclusionBehavioral conditioning inhibits airway allergic inflammation and collagen deposition in murine models of chronic asthma,which is concordant with the effect of actual drug administration.