Functional Study Of Ppm1a In The Maintenance Of Vascular Homeostasis

Maintaining vascular homeostasis is the basis for survival of all higher organisms.Blood vessels form extensive networks that allow the delivery of nutrient to all the tissues as well as the extrusion of toxins.Vascular disease is the leading cause of death worldwide.The abnormal structure and function of blood vessels will eventually progress to vascular diseases,which is the major causes of mortality for patients.To explore the genetic mechanism is crucial for the development of new therapies for diseases with vascular abnormalities.Despite the identification of several signaling pathways involved in the vascular diseases,the underlying molecular mechanisms is still largely obscure.To study the mechanism of vascular development and maintenance is of realistic significance for the diagnosis and treatment of vascular diseases.Protein phosphatase magnesium-dependent 1A(Ppm1a)is the member of the serine/threonine protein phosphatase PPM family with magnesium/manganese-dependent monomeric phosphatase.Transforming growth factor-?(TGF-?)signaling pathway plays an important role in vascular development and homeostasis.Members of the TGF-? superfamily such as TGF?1,TGF?R2,ALK1,ALK5 and intracellular mediator Smads were absent in genetically engineered mice,which would lead to decrease of TGF-? signaling activities and severe defects in vascular development.However,the effect of activated TGF-? signaling in vascular development and maintenance in vivo is not largely clear.Ppm1 a can dephosphorylate a variety of proteins including Smads and P38,thus playing an inhibitory role in TGF-? and BMP signaling pathway.whether Ppm1 a is required for development and maintenance of blood vessels through hyperactivation of TGF-? signaling within endothelial cells still remains largely unknown.In this study,we explored the function of Ppm1 a in vascular development and homeostasis,by making use of endothelial-specific Ppm1 a gene knockout mice.To generate endothelial-specific Ppm1 a knockout mice,Tie2-Cre transgenic mice were mated with Ppm1 a conditional knockout mice(Ppm1afl/fl).The Tie2-Cre;Ppm1afl/fl mice were viable.Immunohistochemistry revealed that there were not structural abnormalities of blood vessels of the organs,including the brain,aorta,kidney and lung,were observed as compared to control mice.Glycoprotein endothelial cell membrane-bound mucin(Endomucin)is a marker of endothelial cells.Previous studies have shown that Endomucin is mainly expressed in the vein,with limited expression within the aorta intima of embryos.A primary function of the endomucin is that abrogation on quiescent endothelial cells enables neutrophils to adhere firmly.Downregulation of endomucin is of vital importance to facilitate adhesion of blood leukocytes into inflamed tissues.Immunohistochemical staining of endomucin showed no evident differences between in wild type control and mutant blood vessels at postnatal day 7(P7).But in mutant mouse at 1 month,endomucin emerged within aorta.Moreover,endomucin expression is constant,which revealed that endomucin expression is acquired.Interestingly,we found that phospho-Smad1/5/8 was significantly upregulated within the endothelial cells in mutant mice.These observations provided the genetic evidence that Ppm1 a could suppress the activity of Smad1/5/8.Therefore,what is the relationship between the Smad1/5/8 activation and endomucin acquired expression.It is unknown whether TGF-? regulates endomucin acquired expression in vascular endothelial cells.The significance of acquired expression of endomucin in adult aorta is not clear and needs further study.Many studies have pointed to that endothelial mesenchymal transition(EndMT)can be induced the abnormal activation of TGF-? signal,which plays an important role in the onset and progression of vascular disease.Because the TGF-? signaling pathway is a major inducer of EndMT,we investigated whether the EndMT switch in endothelial-specific Ppm1 a gene knockout mice.We examined some tissues including the kidney,lung,aorta,brain in mutant mice.However,as compared with in control mice,there were not significant fibrosis in mutant mice by Masson staining.These results were also confirmed under either hypoxia or low pressure conditions.Blood-brain barrier is a special vascular barrier structure,which can accurately control the import and export of a variety of substances in central nervous system and peripheral blood,so as to maintain the homeostasis of the central nervous system microenvironment.It is of significance to maintain the integrity of the central nervous system.TGF-? signaling pathway plays an important role in the maturation of the blood-brain barrier.We also examined the permeability of blood-brain barrier in endothelial-specific Ppm1 a gene knockout mice.However,blood-brain barrier(BBB)permeability did not be increased.According to some reports,reducing oxygenation,or hypoxia is a characteristic of many brain diseases and a major stress factor that significantly increases blood-brain barrier permeability.As the same as the above result,blood-brain barrier permeability did not be increased under hypoxia and hypobaric stress conditions.In summary,our study found that Ppm1 a deficiency in endothelial cells had no significant effect on the vascular structure of the brain,kidney,liver,heart tissues in adult,except for the acquired expression of endomucin in the endothelial cells of adult aorta as well as the up-regulation of phospho-Smad1/5/8.This study firstly described the physiological function of Ppm1 a in vascular development and maintenance,and provide the genetic basis for the new understanding of vascular development and the maintenance of vascular integrity.The transforming growth factor-?(TGF-?)and Notch pathway both play critical roles in vascular development and pathogenesis of vascular diseases.Cross-talk between the TGF-? and Notch pathways in cultured endothelial cells has been reported.Furthermore,endothelial TGF-?/Smad4 signaling cooperates with the Notch pathway to maintain cerebrovascular integrity.TGF-? signaling could modulate Notch pathway at multi-levels.Previously we have found a remarkably decreased Notch signaling activity in Smad4-deficient brain endothelial cells.However,whether Notch receptors serve as direct downstream targets of TGF-?/Smad4 signaling in endothelial cells is by far unknown.Here we show that TGF-?1 and bone morphogenetic protein 4(BMP4)treatments increase the expression of both Notch1 and Notch4 at transcriptional level.In addition,Smad4 physically associates with the Smad binding sites on the Notch4 promoter,which is largely enhanced under the treatment of TGF-?1 and BMP4.Importantly,TGF-?1 and BMP4 fail to transactivate Notch4 in the absence of endogenous Smad4 or the Smad binding regions on the Notch4 promoter.Hence,our findings reveal,for the first time,that the expression of Notch receptor could be directly up-regulated by Smad4-mediated TGF-?/BMP signaling in cerebrovascular endothelial cells.