| [Background]The incidence of diabetes increased year by year,the biggest threat to the patients with diabetes comes from its complications.Diabetic wounds,one of these serious complications,have threatened to the survival and quality of life of diabetic patients with characteristics of difficult to heal,susceptible to infection and high amputation rate.Diabetic wound has become one of those medical problems to be solved urgently.In 2010,an article published on "New England Journal of Medicine"reported that China is now entering a period of high incidence of diabetic ulcers,and prevalence of diabetes in adults over the age of 20 was 9.7%(92.4 million diabetics),the prevalence of pre-diabetes 15.5%(14.82 million pre-diabetic patients)[1].The mechanisms underlying delayed diabetic wound healing is complex,the molecular mechanisms have not been fully elucidated.At present,clinical treatment commonly used is still confined to conventional therapy,the effect is not ideal.Therefore,how to effectively promote wound healing in diabetes has become a multi-disciplinary issue with common concern.It is still need new strategies and breakthrough to overcome the diabetic wounds.Normal wound healing is a complex and orderly process with three phases,including inflammation,proliferation and modeling phase.A variety of cells,cytokines and extracellular matrix and other factors are involved in.Differentiation-related growth factors such as fibroblast growth factor(FGF),epidermal growth factor(EGF),nerve growth factor(NGF),platelet-derived growth factor(PDGF)regulate repair cell activation and proliferation and promote formation of a large number of capillaries.These repair cell include fibroblasts,endothelial cells,keratinocytes and so on.New blood capillaries,collagen secreted by fibroblasts and extracellular matrix constitute granulation tissue,while keratinocytes proliferate to cover the wound,followed by remodeling of granulation tissue.Thus,the process of wound healing is complete.Among them,the formation of the wound angiogenesis and granulation tissue in wound healing is the key process.Endothelial cell proliferation,differentiation,migration and eventually fused into a primary vascular network,which then enters the primary vascular network remodeling process:budding,branching,growth in different directions,recruitment and other supporting cells eventually develop into mature vascular system[2].In the case of a sufficient number of endothelial cells exist,being assembled into functional vessels is more meaningful for revascularization and wound healing process.Skin of patients with diabetes is vulnerable to injury.The diabetic wounds are often recurrent with delayed healing and easy to formation of skin ulcers(non-healing wound)after injury.Diabetic wound healing process is disorder,especially inflammation and proliferation phases[3,4].Currently impaired wound healing mechanism in diabetes is still unclear and generally considered to be the result of many factors.Delayed wound healing in diabetes mainly due to blood vessels,nerves and metabolic multiple factors.Among them,the diabetic vascular disease in skin tissue,impaired angiogenesis in wounds,the disordered formation of granulation tissue,may be important causes of impaired wound healing in diabetes[5].Poor vascularization and vascular insufficiency are pathological features of diabetic woundsc[6].Clinically,lack of fresh wound granulation tissue is often observed in diabetic wounds[7].Angiogenesis is the main component of granulation tissue,providing nutrition and transport of metabolites to the wound area and play an important role in the wound healing process[8].Lack of new blood vessels,may lead to impaired wound healing or healing.Therefore,after the formation of wound angiogenesis disorders has challenged the further research.Related study[9]showed that vascular endothelial cells with insufficient quantity or dysfunction in skin wounds with diabetes is related to their decreased proliferation and increase apoptosis.Meanwhile,a study on the expression of growth factor and angiogenesis in endothelial cells of type 2 diabetic rats showed that the in vitro ability of angiogenesis of cardiac microvascular endothelial cells in diabetic rat is decreased.Reduced VEGF and its receptors expression caused by post-transcriptional abnormal may result in impaired neovascularization in diabetes.Therefore,the systematic study of targeting angiogenesis in diabetic wounds will contribute to the development of new treatments for wound healing in diabetes[10].At the molecular level,growth factors modulate cellular activity by signaling pathways during the wound healing process.Studies have shown that hypoxia-inducible factor-1(HIF-1)in the wound healing process has played a key role.As a key transcription factor in hypoxic regulation,HIF-1 has shown to regulate and control over more than 100 target genes so far,including angiogenesis-related factor(VEGF,ANG-1,ANG-2,VEGFR,IGF-2),energy metabolism-related factor(ALDA,GLUT-1,GLUT-3,LDHA,PFK1,ENO1),cell proliferation factor(cyclin D1)and so on(see Table 1).After the formation of the wound and hypoxic environment developed locally,HIF-1 regulates the expression of target genes and effect proliferation,differentiation,senescence,apoptosis of repair cells,promote tissue vascularization[11,12].HIF-1 is a heterodimer composed of the HIF-la and HIF-1(3 subunits.Physiological activity of HIF-1 is determined by expression of HIF-la subunit.Under normoxia,HIF-la expression remained at a low level and ease of ubiquinone by proteolytic.Under hypoxia,the transcription and translation levels of HIF-1? are increased exponentially.It is combined with HIF-1? into dimers and form active HIF-1 which can be resistant to proteolytic enzyme degradation active.HIF-1 has claimed the role of the master player that orchestrates the cellular responses to oxygen deprivation.Moreover,HIF-1 is activated or influenced through oxygen-independent mechanisms via growth factors,deregulated oncogenes,and/or tumor suppressors.Whereas the HIF prolyl hydroxylases(PHDs)regulate HIF-1 during hypoxia,the PI3K,Akt and MAPK pathways primarily mediate non-hypoxic HIF regulation[13](Figure 2-1).As demonstrated by an increasing number of studies,HIF-la contributes to angiogenesis during wound healing[14].However,glucose and hyperglycemia have been shown to impair HIF-la stability and function[15,16].A growing body of evidence suggests that HIF-1 also affects the reepithelialization of the wound bed by increasing keratinocyte migration while decreasing their proliferation.Diminished HIF-1 levels and activity have been documented in conditions of impaired wound healing,such as wound healing in aged and diabetic mice[11,17].Furthermore,previous studies have revealed that impaired HIF-1 and VEGF expression in the skin of the limbs leads to decreased vascular vessel density,poor blood circulation and impaired wound healing,which are important causes of diabetic foot injury[18-2].HIF-1? gene therapy showed synergistic effects with combined therapy on tissue perfusion in a mouse model of limb ischemia[20].However,the molecular mechanisms underlying this impaired HIF-1 signaling and whether AIP1 plays a role in this process are not known.Apoptosis signal-regulating kinase 1-interacting(ASK1-interacting)protein-1(AIP1),also known as DAB2-interacting protein(DAB2IP),is a member of the Ras GTPase-activating protein family that has been implicated in cell growth inhibition and apoptosis[21.22].AIP1 is recruited to the VEGFR2-PI3K complex,where it binds to both VEGFR2 and PI3K p85 during the late phase of the VEGF response;in turn,this binding leads to the inhibition of VEGFR2 signaling.Given the critical role of VEGFR2 signaling in angiogenesis,regulation of VEGFR2 activity/activation may represent an important mechanism for its control[23].Consistent with its role as an inhibitor of cell survival,growth and angiogenesis,AIP1 expression is often downregulated in various human cancers[21,24,25].These data suggest that AIPI may function as a tumor suppressor.However,the role of AIP1 in the molecular mechanisms behind diabetic wound healing are unclear,and the effects of high glucose and insulin on AIPI gene expression are unknown.Vascular endothelial growth factor(VEGF),also known as vascular permeability factor(VPF),is an endothelial cell-specific mitogen,but also an effective inducing factor to promote angiogenesis,increase vascular permeability and chemotaxis of endothelial cells[26-29].According to the different RNA splicing,VEGF has nine types of isoforms,of which VEGF165 is the main secreted form.VEGF 165 is also a major effector molecules with strongest effect on proliferation and angiogenesis of endothelial cells[30-31].After binding the tyrosine kinases receptor VEGFR on vascular endothelial cells,VEGF activates different signaling pathways which play a series of biological effects.To date,it has been found the three kinds of VEGF receptors(VEGFRs):VEGFR-1,VEGFR-2 and VEGFR-3.As the main receptor of VEGF,VEGFR-2 is mainly in vascular endothelial cells,also located in the hematopoietic stem cells,macrophages,and so on.Its main role is to regulate the proliferation,survival,migration,and changes in permeability of endothelial cells[27,28].Studies have shown that VEGFR-2 by activating PI3K/Akt regulate endothelial cell survival,migration[32,33].In addition,VEGF activates endothelial nitric oxide synthase(eNOS)to generate NO via PI3K/Akt pathway after binding to VEGFR-2,which result in changes in vascular permeability[34].PI3K/Akt pathway is widely existing in the cells and subject to activation of cytokines.It involved in cell proliferation,differentiation,apoptosis,and glucose transport regulation and other cellular functions.Akt also known as protein kinase B or PKB,a serine/threonine protein kinases,is a proto-oncogene and a major downstream effector of growth factor signaling.Akt has a wide array of pro-growth,anti-apoptotic effects when activated by growth factors through PI3K[35].Studies have demonstrated that PI3K/Akt signaling is required for VEGF expression through HIF-1 in response to growth factor stimulation and oncogene activation[36-40].Hypoxia activates nuclear factor ?B(NF ?B)via PI3K/Akt signaling and its subunits can bind to HIF-1? promoter which leads to increased expression of HIF-1?mRNA[41].The use of chemical inhibitors of PKC and PI3K/Akt signaling pathway experiments found that:These two signals are mutually reinforcing so as participate in the protection HIF-1? from degradation by pVHL which results in increased expression of HIF-1?[42,43].In addition,positive role of PI3K/Akt on HIF-1? activity has also been confirmed in hepatoma cells[441 and breast cancer cells[45].As mentioned earlier,AIP1 binding to PI3K p85 and VEGFR2 formed VEGFR2-PI3K complex,inhibiting VEGFR2 signaling pathways.However,the relation between AIP1 and PI3K/Akt/HIF-1 signaling pathway during diabetic wound healing process has not been elucidated.In summary,HIF-1 and downstreams expression levels and activity closely correlated with angionesis during the process of wound healing.Glucose and hyperglycemia reduce HIF-1 signaling expression levels and activity.However,the molecular mechanism underlying it is still not clear.The impaired HIF-1 signaling is an important mechanism for the delayed diabetic wound healing,whether this impaired signaling is related to AIP1 or a result of AIP1 regulation?At present,the effect of high glucose/high insulin on AIP1 expression and the role of it during diabetic skin wound healing process have not been reported in the world.Is AIP1 an important molecule involved in the regulation of this pathological process?Therefore,we performed our experiments in vitro and in vivo from October 2013 to December 2014.To define the relationship between AIP1 and PI3K/Akt/HIF-1?signaling in diabetes,we investigated changes in AIP1 expression and HIF-1?signaling in cells exposed to high glucose,high insulin,and their combination(high glucose+high insulin),simulating conditions observed during the progression of type 2 diabetes.We hypothesized that the inhibition of HIF-1? signaling activity in human umbilical vein endothelial cells in diabetes patients is a result of the regulation of AIP-1.Angiogenic disorders are key factors in delayed wound healing in diabetes,and decreased HIF-1? expression is involved in these disorders.Therefore,we propose that AIP1 expression is altered under high glucose and insulin conditions.These alterations affect the HIF-1 signaling pathways via PI3K/Akt,resulting in impaired angiogenesis in diabetic wound healing.We established BALB/c model for the study of diabetic wounds and try local injection into wound margin with recombinant adenovirus containing AIP1 siRNA.Wound healing rate,microvessel density in tissue,the amount of collagen deposition were observed.We attempt to explore the molecular mechanisms of diabetic delayed wound healing and to provide new ideas for the clinical targeted treatment of diabetic wounds.[Aims]The goal of this study was to explore the role of AIP1 in the mechanism underlying delayed diabetic wound healing and possible relationship with PI3K/Akt/HIF-1?signaling.We also attempt to find a new target and gene therapy for diabetic wounds treatment.[Methods]We investigated expression of AIP1 and PI3K/Akt/HIF-1? signaling in human umbilical vein endothelial cells at mRNA and protein level by 30 mmol/1 glucose(high glucose,HG),1 nmol/1 insulin(high insulin,HI),and their combination(high glucose/high insulin,HG/HI),simulating conditions observed during progression of type 2 diabetes.The expression of AIP1,HIF-1? and VEGF of the wounds in T2DM and normal mice were detected by real-time PCR and western blot during wound healing.We detected changes in HIF-1? and VEGF expression with AIP1 siRNA interference by real-time PCR and western blot.Then,we used recombinant adenovirus containing AIP1 shRNA to treat the wounds in T2DM mice.AIP1,HIF-1? and VEGF expression were observed by real-time PCR and western blot.Protein levels of CD31 were detected using immunohistochemistry,collagen staining was used with VG method.Statistical analysis was performed using SPSS software(version 19.0,SPSS Inc.,Chicago,IL,USA).The results are presented as the means± standard deviations(x±s).Differences in means between groups were analyzed for significance using Student's t-test or One-Way ANOVA as appropriate,A P-value<0.05 was considered significant.[Results]Compared with 5.5 mmol/1 glucose and no insulin(control),high glucose alone,increased AIP1 expression at 24 h in mRNA and protein levels and decreased PI3K/Akt/HIF-1? expression at mRNA and protein level.Insulin can reverse the effects of HG on gene expression partially.Knockdown of AIP1 significantly increase HIF-1? and VEGF expression both at mRNA and protein level in HUVEC.AIP1 expression is also decreased in wounds of T2DM mice during wound healing.In comparison with controls,local injection of Ad-AIP1-siRNA into wound beds and margins increased HIF-1? and VEGF expression and promoted angiogenesis,collagen deposition and the wound healing rate in T2BM mice.[Conclusion]High glucose and high glycemia increase AIP1 expression and insulin can reverse the effect of high glucose on genes expression partially.AIP1 may play an important role in molecular mechanisms of delayed wound healing in diabetes.Deficiency of PI3K/Akt/HIF-la signaling regulated by increased AIP1 compromises skin wound healing in diabetic mice.Therapeutic potential of AIP1 targeting in delayed diabetic wound healing may be a new hope for diabetic wound treatment. |
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