The Mechanism Research Of VEGFR3 Activates The Neural Stem Cells Potential Of Pkd2l1~+CSF-cNs Via PI3K/Akt Signaling Pathway

Objective:Many neuronal defects caused by spinal cord injury(SCI)are challenging to repair,and neurological functions cannot be restored.Neural stem cells(NSCs)can proliferate and differentiate into neurons and oligodendrocytes,thus repairing spinal cord damage.Neuronal stem cells in the central canal of the spinal cord are Pkd2l1~+cerebrospinal fluid-contacting neurons(CSF-cNs).Under normal conditions,these cells are resting,and it is urgent to determine how to activate the neural stem cell potential of Pkd2l1~+CSF-cNs.It was discovered that VEGFR3 plays an essential role in neural stem cells.VEGFR3 has been shown to act on neural progenitor cells in developing African Xenopus and mouse brains to stimulate neural progenitor cell proliferation.Studies on the specific function and mechanism of VEGFR3 in Pkd2l1~+CSF-cNs will help us better understand the neural stem cell properties of Pkd2l1~+CSF-cNs and the therapeutic potential for spinal cord injuries.Methods:This study investigated the role of VEGFR3 in activating the neural stem cell potential of Pkd2l1~+CSF-cNs using a combination of animal experiments,cellular experiments,molecular biology experiments,and bioinformatics.1.Immunofluorescence was used to determine the expression of the neural stem cell markers Nestin and Sox2 and the juvenile neuronal marker Dcx on Pkd2l1~+CSF-cNs in vivo.Pkd2l1~+CSF-cNs were purified by flow cytometry and lentiviral screening.We performed the neuro-spheres assay of these cells in vitro.Next,the expression of Nestin,Sox2,and Pkd2l1 was detected by Western blot and q RT-PCR on NE-4C,neural stem cells of lateral ventricular,and Pkd2l1~+CSF-cNs;By performing differentiation assays on Pkd2l1~+CSF-cNs,the capacity for differentiation of these cells was determined.2.After trans-lateral ventricular injection of VEGF+b FGF,the expression of Pkd2l1 was detected by q RT-PCR.The co-expression of Pkd2l1 with neural stem cell markers(GFAP,Nestin),proliferation markers(Ed U,Ki67),and immature neuronal markers(Dcx)was detected using immunofluorescence.After VEGF+b FGF injection,q RT-PCR was used to detect the expression of Nestin,GFAP,Ki67,and Dcx.Nissl staining revealed neuronal activity in the spinal cord’s central canal.3.Spinal cord injury models were created,and spinal cord tissues(including the sham group)were collected at 1,3,7,and 14 days after SCI.The co-expression of Pkd2l1 with neural stem cell markers(GFAP,Nestin),proliferation markers(Ed U,ki67),and immature neuronal markers(Dcx)was detected by immunofluorescence at3 days after SCI;Then,the expression of Nestin,GFAP,Ki67,and Dcx was detected by q RT-PCR 3 days after SCI;Neuronal activity in the central canal of the spinal cord was detected using Nissl staining.4.Spinal cord tissue was collected 3 and 7 days after SCI.Western blot and q RT-PCR were used to detect the expression of VEGFR3;The immunofluorescence assay was conducted to investigate the expression of VEGFR3 on mouse tissues at 7days after SCI.5.Cellular immunofluorescence of cultured CSF-cNs revealed differences in VEGFR1,VEGFR2,and VEGFR3 in CSF-cNs;Tissue immuno-fluorescence of mouse spinal cord tissues revealed the co-expression of VEGFR3 with biomarkers including Pkd2l1,GFAP,and Foxj1.The cultured CSF-cNs and neural stem cell line(NE-4C)were then subjected to detect the co-expression of VEGFR3,Ascl1,and Pkd2l1 using immunofluorescence,as well as Western blot and q RT-PCR.6.We observed the proliferation of CSF-cNs by knock-down or overexpressing VEGFR3 in CSF-cNs using neurosphere-forming assay,Ed U assay,immuno-fluorescence,and CCK8 assay.The expression of Nestin,Ascl1,and Egfr was also detected Using Western blot.Following VEGFR3 knockdown or overexpression.,Caspase3 and Pro-Caspase3 expression levels in CSF-cNs were evaluated by Western blotting.The Tunel assay was used to characterize apoptotic cells in CSF-cNs following either knockdown or overexpression of VEGFR3.Immunofluorescence was used to assess the expression of Caspase3 in CSF-cNs following modulation of VEGFR3 expression via knockdown or overexpression.7.Transcriptome and proteome sequencing were used to examine the downstream pathway modifications after VEGFR3 knock-down;Western blot was used to explore the expression of PI3K,p-PI3K,Akt,and p-Akt;Following the knock-down of VEGFR3,the Akt pathway activator SC79 was introduced to the CSF-cNs.8.Using the VEGFR3 inhibitor SAR131675 and VEGFR3 agonist VEGF-C,immunofluorescence was used to detect the co-expression of the resting NSCs markers(Cd133,GFAP),activating NSCs markers(Egfr,Nestin),Ed U,Ascl1,and Pkd2l1.Multiplex immunohistochemistry was used to identify the multiple-labeled Cd133,GFAP,and Pkd2l1.Finally,after the injection of VEGF-C and SAR131675,Nissl staining was used to determine the activity of neurons in the spinal cord’s central canal.Results:1.The Pkd2l1~+CSF-cNs exhibited expression of neural stem cell markers Nestin and Sox2,as well as the immature neuronal marker Dcx.When cultured in vitro,these CSF-cNs displayed green fluorescence under a fluorescence microscope,and a substantial number of neurospheres formed within one week.Western blot and q RT-PCR analyses revealed similar expression patterns of neural stem cell genes(Sox2 and Nestin)across all three cell types.Notably,CSF-cNs expressed Pkd2l1 at elevated levels compared to the other two neural stem cells(P<0.001),which exhibited low expression.Following a seven-day induction period for cell differentiation,these CSF-cNs were capable of differentiating into Neun-positive neurons,S100β-positive astrocytes,and Olig2-positive oligodendrocytes.2.After injecting VEGF+b FGF or an equal volume of PBS into the right ventricle of mice for one week,q RT-PCR showed that Pkd2l1 expression was significantly higher in the lateral ventricle injection of VEGF and b FGF than in the PBS group(P<0.05).Pkd2l1~+Nestin~+co-expressing cells were significantly increased after VEGF+b FGF injection compared to the PBS group(P<0.0001).q RT-PCR analysis showed that the Nestin expression was significantly increased after VEGF and b FGF injection(P<0.05).In addition,Pkd2l1~+GFAP~+co-labeled cells were also considerably increased after VEGF and b FGF injections(P<0.05).q RT-PCR analysis showed that GFAP expression was significantly increased after VEGF and b FGF injections(P<0.05).Notably,VEGF+b FGF treatment induced more Pkd2l1~+Ki-67~+co-expressing cells and Pkd2l1~+Dcx~+immature neurons(P<0.0001).Furthermore,the proliferation of CSF-cNs was also confirmed in the Pkd2l1~+Ed U~+co-expressing cells in the VEGF+b FGF group(P<0.01),and Nissl staining showed a significant increase in the number of neurons(P<0.05).q RT-PCR analysis revealed substantial differences in Ki-67 and Dcx expression after VEGF and b FGF injection(P<0.05).3.The expression of Nestin at different time points(1,3,7,14 days)after SCI was verified using q RT-PCR.The results showed that Nestin expression peaked at 3 days after SCI(P<0.0001).q RT-PCR results showed a significant increase in Pkd2l1expression(P<0.05).The molecular responses of Pkd2l1~+cells to the NSC markers Nestin and GFAP were examined on day 3 after SCI.The percentage of Pkd2l1~+Nestin~+co-expressing cells increased significantly after spinal cord injury(P<0.05).q RT-PCR analysis showed a significant increase in Nestin expression after SCI(P<0.0001).Similarly,the density of Pkd2l1~+GFAP~+co-expressing cells was significantly higher in the spinal cord injury group than in the sham group(P<0.01).There were fewer co-labeled cells in the sham-operated group.q RT-PCR results showed a significant increase in GFAP expression after spinal cord injury(P<0.0001).The percentage of Pkd2l1~+Ki-67~+and Pkd2l1~+Dcx~+co-expressing cells was significantly higher in the spinal cord injury group than in the sham group(P<0.05).Ed U incorporation experiments showed that Pkd2l1 positive cells began to proliferate after SCI,and Nissl staining also showed an increase in the number of neurons(P<0.05).q RT-PCR analysis showed a significant increase in Ki-67 and Dcx expression after spinal cord injury(P<0.01).4.Spinal cord tissue from the 3-day and 7-day post-SCI and sham group was analyzed using Western blot.The results indicated that VEGFR1,VEGFR2,and VEGFR3 were up-regulated following SCI,with the highest expression occurring seven days after SCI(P<0.01).However,among the three VEGF receptors,the expression of VEGFR3 was the most prominent.The q RT-PCR results were comparable to the Western blot results.The percentage of Pkd2l1~+Ascl1~+and Pkd2l1~+VEGFR3~+cells was more significant in the SCI group than in the sham group(P<0.05),indicating the expression of VEGFR3 and Ascl1 increased.5.In vitro cellular immunofluorescence revealed that VEGFR3 expression was highest in CSF-cNs formed neurospheres.In addition to CSF-cNs,the central canal of the spinal cord contains astrocytes(GFAP),neurons(Neun),and ependymal cells(Foxj1).Immunofluorescence analysis revealed that VEGFR3 was predominantly expressed in CSF-cNs.Western blot results also showed that CSF-cNs could express Pkd2l1,Ascl1,and VEGFR3,whereas NE-4C cannot express Ascl1 and Pkd2l1.The expression of VEGFR3 was slightly more significant than that of CSF-cNs.The q RT-PCR results were comparable to the Western blot results.6.The q RT-PCR and Western blot were used to examine the effects of VEGFR3manipulation in CSF-cNs with overexpressing and interfering VEGFR3 lentiviruses.The results revealed that both knockdown and overexpression of VEGFR3 had significantly different VEGFR3 expression levels than the control group(P<0.0001).It was demonstrated that overexpression of VEGFR3 could promote the proliferation of CSF-cNs-formed neurospheres.The neurosphere formation was smaller in the interfered group,and the percentage of Ed U-positive cells was lower than in the control group(P<0.05).After overexpression of VEGFR3,the number of Phh3-positive cells was higher than in the control group(P<0.01).The CCK8 assay further confirmed that the growth rate of CSF-cNs increased after overexpression of VEGFR3,while the interference group remained at a lower level(P<0.05).The Western blot results indicated that the VEGFR3 overexpression group expressed more Ascl1,Egfr,and Nestin than the control group.Western blot analysis revealed that the expression of Caspase3 and Pro-Caspase3 was higher in the VEGFR3 interference group than in the control group.There was no significant difference between the number of apoptotic cells in the VEGFR3 overexpression and control groups.The immunofluorescence results demonstrated that the number of Caspase3-positive cells in the VEGFR3-interfered group was significantly greater than in the control group(P<0.01).In contrast,there was no significant difference between the control and VEGFR3-overexpressed groups.Thus,the preceding results confirmed that inhibiting VEGFR3 promoted apoptosis of CSF-cNs.7.Following VEGFR3 interference,transcriptome sequencing data unveiled 132up-regulated genes and 707 down-regulated genes compared to the interfering groups.KEGG pathway enrichment analysis indicated that the PI3K/Akt pathway was most significantly down-regulated.Proteome sequencing data identified 334 highly expressed genes and 236 genes with low expression levels.KEGG pathway enrichment analysis revealed that the PI3K-Akt signaling pathway was considerably inhibited after VEGFR3 interference.These findings suggest that VEGFR3 may potentially promote CSF-cNs activation via the PI3K/Akt pathway.Western blot analysis revealed that the expression of phosphorylated Akt and PI3K were higher than in the control group.Similarly,Compared to the control group,the interference group had lower levels of phosphorylated Akt and phosphorylated PI3K.When a Akt pathway agonist was added to the VEGFR3-interference group,it was demonstrated that the expression of p-Akt was rescued.8.By lateral ventricular injection of the VEGFR3 activator VEGF-C,Immunofluorescence labeling demonstrated a significant decrease in the proportion of Pkd2l1~+Cd133~+and Pkd2l1~+GFAP~+CSF-cNs in the activator group compared to the control group(P<0.05).The results of multiple immunohistochemistry assays indicated that the percentage of Pkd2l1~+Cd133~+GFAP~+CSF-cNs in the activator group was significantly lower compared to the control group.q RT-PCR analysis showed a significant reduction in Cd133 and GFAP expression after injection of VEGF-C(P<0.05).The ratios of Pkd2l1~+Nestin~+and Pkd2l1~+Egfr~+CSF-cNs were significantly higher in the activator group compared to the control group(P<0.01).q RT-PCR analysis showed a significant increase in Egfr and Nestin expression after injection of VEGF-C(P<0.001).For multiplex immunohistochemistry,the proportion of Pkd2l1~+Nestin~+Egfr~+CSF-cNs was also markedly higher in the inhibitor group compared to the control group.Immunofluorescence results showed a significantly higher proportion of Pkd2l1~+Ascl1~+CSF-cNs in the activator group compared to the control group(P<0.01).Immunofluorescence results also showed that the proportion of Pkd2l1~+Ed U~+CSF-cNs was considerably higher in the activator group than in the control group(P<0.01).Nissl staining showed a significantly higher number of Nissl bodies in the activator group than in the control group(P<0.01).9.After intraperitoneal injection of the VEGFR3 inhibitor SAR131675,immunofluorescence revealed that the inhibitor group had a significantly higher proportion of Pkd2l1~+Cd133~+and Pkd2l1~+GFAP~+CSF-cNs than the control group(P<0.0001).The results showed a significant increase in Cd133 and GFAP expression after injection of SAR131675 by q RT-PCR analysis(P<0.0001).For multiplex immunohistochemistry,the inhibitor group also had a higher proportion of Pkd2l1~+Cd133~+GFAP~+CSF-cNs than the control group.Moreover,the results of immunofluorescence labeling demonstrated a decreased proportion of Pkd2l1~+Egfr~+CSF-cNs in the inhibitor group relative to the control group,as well as a significant reduction in the proportion of Pkd2l1~+Nestin~+CSF-cNs(P<0.0001).q RT-PCR analysis further corroborated these findings,revealing lower expression levels of Egfr and Nestin in the inhibitor group compared to the control group(P<0.001).Furthermore,multiple immunohistochemistry assays indicated a significant decrease in the proportion of Pkd2l1~+Nestin~+Egfr~+CSF-cNs in the inhibitor group relative to the control group.Immunofluorescence showed no significant difference in the proportion of Pkd2l1~+Ascl1~+and Pkd2l1~+Ed U~+CSF-cNs between the two groups.Nissl staining showed that the number of Nissl bodies was significantly lower in the inhibitor group compared to the control group(P<0.01).Conclusions:Pkd2l1~+CSF-cNs had the properties of neural stem cells in vivo;VEGFR3 was expressed in Pkd2l1~+CSF-cNs,and VEGFR3 promoted Pkd2l1~+CSF-cNs proliferation;Activation of the PI3K/Akt pathway by VEGFR3 enhanced the proliferation of Pkd2l1~+CSF-cNs.