Renal Targeted Homing Of Hepatocyte Growth Factor Modified Bone Mesenchymal Stem Cells On The Improvement Of Renal Fibrosis Induced By Microbbuble Mediated Ultrasound

Background:Tubulointerstitial fibrosis is the final stage of most chronic nephropathy, andtubulointerstitial fibrosis control or reversing is essential in the therapy of chronic kidneydiseases.Bone marrow stromal cells (BMSCs) have numerous advantages for cell therapy andgene therapy in tissue repair and organ regeneration, including their high plasticity and easeof differentiation. For kidney, BMSCs can differentiate into variety of renal cell, includingmesangial cells, endothelial cells, podocytes and tubular epithelial cells, which suggestBMSCs to be the most attractive cells for the repair of injury renal tissue. BMSCs can alsoprotect the injury kidney which caused by ischemia or reperfusion in the way of theparacrine which can significantly reduced the necrosis of the adjacent cells. However, somestudies showed the poor therapy effect of the simple BMSCs transplantation for thetreatment of injury fibrosis renal tissue. The reason may be related to the way of BMSCstransplantation, the small number of homing cells, the lack of hepatocyte growth factor(HGF) which can significantly improve renal interstitial fibrosis and etc.Recently the application of ultrasound contrast agents (microbubbles) has beenexpanded from diagnosis to treatment. Studies have showed microbubbles (MB) mediatedultrasound (US) engender the ultrasonic cavitation can be used as a gene therapy non-viralvector. Ultrasound-targeted microbubble destruction (UTMD) mediated drug targeteddelivery and gene transfection is recognized as an effective and promising method in recentyears. This study tried to use the characteristic of MB combined with US to modify BMSCs*The study was fund by the National Natural Science Foundation (NO.81071160) with HGF in the optimized acoustic parameters and use the method of UTMD to transplantthe cells in the animal models of renal fibrosis for the gene and cell combination therapy. Inaddition, UTMD can change the microenvironment of the target organ, enhance themicrovascular permeability in the target organ, increase endothelial cell gap, promote thesecretion of cytokine. So all of these changes can create the conditions for BMSCs“homing” the target tissue.So US combined with MB induced the HGF modified BMSCs(HGF-BMSCs) transplantation may promote HGF-BMSCs targeted homing andaggregation to the damaged kidney. In addition this method may result to improve thetherapy efficacy and the improvement of renal fibrosis. The study tested the feasibility,safety and targeting of this medthod and trid to explore its mechanism.Objective：1. To study the effectiveness, feasibility and mechanisms of the MB mediatedtreatment ultrasound combined polyethyleneimine (PEI) increased the transfectionefficiency of BMSCs modified by HGF. An attempt was made to find a non-viral method toachieve high-transfected BMSCs in order to avoid the high toxicity and immunogenicity ofviral vectors.2. To stuy the changes of renal interstitial capillary permeability induced by MBmediated the certain parameters diagnostic ultrasound(DUS) as well as renal morphologyand function.3. To study the feasibility of intravenous BMSCs transplantation which was inducedby MB mediated DUS and was promoted its targeted homing to the impaired kidney ofunilateral ureteral obstruction model.4. To study the effectiveness, feasibility, safety and possible mechanisms of theimprovement of the renal fibrosis treated by BMSCs transplantation and HGF-BMSCstransplantation non-invasively mediated by DUS and MB in order to explore experimentalfoundation for the renal fibrosis therapy.Methods:1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEIThe adherent culture method was used in the isolation and cultivation of BMSCs. Thegrowth curve, cell cycle, transmission electron microscopy and etc. were detected for the biological characteristics. Flow cytometry was applied to detect the expression of cellsurface marker molecules on the BMSCs.Osteogenic and adipogenes is induction ofBMSCs were performed.The plasmid DNA: pEGFP-HGF was constructed. Eachexperiment was arranged based on the uniform design. After tested the cell densities, thetransfection efficiency and the cell viability, the results of the optimized transfectionparameters (the acoustic intensity, the dosage of MBs, and the exposure time) wereanalyzed by regression analysis ultimately.The cell cycle and differentiation capacity of thetransfected BMSCs were detected.The BMSCs fluorescence expression at the time point of48h and7d after transfection were observed using laser scanning confocal fluorescencemicroscopy.The cell damage caused by UTMD and the cell self-recovery were observedusing scanning electron microscopy (SEM).2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safetyThe left kidney of healthy Sprague–Dawley (SD) rat was insonated by UTMD witheither continuous or intermittent mode for5min.The target organ kidney interstitiumcapillary permeability changes were observed by Evans blue (EB) quantification,hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM).Thepositioning of the DIO labed MB targeted release which was observed by the confocal laserfluorescence microscope. Urinalysis was performed to evaluate the glomerular filtration andthe tubular reabsorption.3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodelThe mode of unilateral ureteral obstruction (UUO) was built and evaluated by imagingtests and pathology. MB mediated DUS improved the HGF-BMSCs vein transplantation onthe7d after modeling. Grouping as follow: HGF-BMSCs transplantation in UUO rat modelgroup (UM group), US+MB+HGF-BMSCs transplantation in UUO rat model group (UUMgroup), HGF-BMSCs transplantation in healthy rat group (CM group), US+MB+HGF-BMSCs transplantation in healthy rat group (CUM group). After48hours of celltransplantation，the green fluorescence-positive cells were observed using the confocal laserfluorescence microscope. The survival implanted cells were counted and statisticalanalysised. The target tissue morphology and the ultrastructure of interstitial capillaries after transplantation were observed by HE staining and TEM. The homing factor SDF-1expression was observed and quantitative analysised using immunohistochemical fordamaged kidney.4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin ratsThe mode of unilateral ureteral obstruction recanalization (RUUO) was built andevaluated by imaging tests and pathology. Stem cell vein transplantation was performed onthe7d after modeling. The unilateral ureteral obstruction was relieved on the14d aftermodeling. Grouping as follow: RUUO group, BMSCs group, US+MB+BMSCs group andUS+MB+HGF-BMSCs group. The four observing time points were7d,14d,21d and28dafter modeling. At each time point, items were detected as follows: Imaging tests was usedto observe the renal morphology. HE staining and Masson staining were used to observe thetubulointerstitial damage and evaluate ratings. The gene expression of hepatocyte growthfactor (HGF), transforming growth factor-β(TGF-β) andα-smooth muscle actin (α-SMA) were detected using real time PCR method. The expression of α-SMA wasdetected by immunohistochemistry method. The protein expression of SDF-1, α-SMAwere detected by western blot method. The blood and urine were collected for the renalfunction detection followed as serum creatinine, blood urea nitrogen and creatinineclearance.Results:1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEIThe identification results showed that specific antigens CD44, CD29, and CD90werepositive expressed and the specific antigens CD34, CD45, and CD11b of other cell lineagewere not expressed in the BMSC cultures. Adipogenic and osteocyte differentiation weresuccessfully induced in BMSCs. The best match of parameters are as follow, Q=0.6W/cm2, MB=106/ml, T=30s. The transfection efficiency (38.81±2.58)%and cellviability (82.10±1.77)%both reached a high level in the optimal parameter. Theexpression of GFP continued for7days after transfection. Comparing the differentinfluencing factors, PEI:DNA+MB+US group was significantly different (p <0.05). By day7after transfection,57.43±1.56%of BMSCs were transfected with MB-mediated US combined with PEI:DNA; the BMSC viability on7days after transfection was78.98±3.11%. The transfected BMSCs still maintained their differentiation capability (G1phase82.93%). With osteogenic induction of transfected BMSCs, there was a significant increasein calcium nodule formation, with red nodules staining positive with alizarin red. Westernblot analysis showed that interest protein HGF was successfully expressed in thepEGFP-HGF group, compared with no expression in the control and pEGFP-N1groups.Tiny holes were seen on the cell membranes. The sonoporation pore sizes were not uniform,and there was distortion on the cell surface. By day7after sonoporation, the BMSCs hadrecovered; most of the tiny ‘‘holes’’ had disappeared, and the cell surface was smooth.2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safetyThe Continuous DUS without MB (CUS) group or Intermittent DUS without MB (IUS)group showed no significant difference compared with control group (P>0.05) using EBquantification. After UTMD, EB content of either continuous DUS with MB group (CUS+MB) group orIntermittent DUS with MB (IUS+MB) group showed significant increasecompared with three controls respectively (P<0.05). The IUStMB post6h group, the EBcontent reached3.15±2.20μg/g which decreased significantly compared with theIUStMB group.The EB content reached0.690±0.540μg/g in the IUStMB post-24hgroup, which was no statistically significant difference between the control group (P>0.05).Pathology detection results suggested the interstitium was damaged and the microvesselwall was broken after UTMD, while the glomerular basement membrane was intact.Theinterstitial capillary injury can self-recovery in24h after UTMD. Dio-labeled MB followedby DUS and observed under fluorescent microscope suggested the MBs are predominantlylocalized in the exposed kidney over other unexposed organs. However, IOD in IUS+MB(IOD=2092.17±342.36) and CUS+MB (IOD=943.48±179.07) showed significantincrease over control group and the right kidney (P<0.05). Furthermore, IOD in IUS+MBgroup showed significant increase compared to CUS+MB group (P<0.05). The urine resultshowed the protein level from (-) up to (++) in normal rats ranges without significantchange.3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodel The green fluorescence positive BMSCs were observed neither in CM group nor inCUM group in healthy rats. However, in UM group and UUM group the green fluorescencepositive BMSCs were observed in model rats. The number of positive cells wererespectively16.41±5.37and86.39±8.49, with significantly difference (P<0.05). HEstaining showed that after UTMD, the interstitium was damaged and the microvessel wallwas broken, lots of red blood cells were leaked, interstitial structure was loose and edema,the renal interstitial permeability increased significantly. The similar results were foundusing TEM. The expression of homing factor SDF-1located in the cytoplasm of the distalconvoluted tubule and the rarely proximal tubule which were detected byimmunohistochemistry. The UUM group (22146.49±1920.73) is not difference highercompared with the UM group (18643.94±2046.29), P>0.05. The CUM group (5528.36±1021.81) was statistically significant difference with the CM group (5162.38±1376.54), P <0.05.4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin ratsThe renal morphology of RUUO rat model in each group was observed usingultrasound imaging teast in different time points (7d,14d，21d，28d after modeling). TheUS+MB+BMSCs group and the US+MB+HGF-BMSCs group were significant differentbetween the RUUO group and the BMSCs group. The renal interstitium pathological scoresshowed that the US+MB+BMSCs group and US+MB+HGF-BMSCs group weresignificantly lower compared with the BMSCs group and the RUUO group (P<0.05) on the21d and28d.In addition,the US+MB+HGF-BMSCs group was significantly between groupson28d (P<0.05). Fibrogenic factor TGF-β in the renal tissue was detected by Real-timePCR on the four time points which suggested that the BMSCs transplantation treatmentgroups were significant different between the RUUO group (P<0.05) after14d. However,the US+MB+BMSCs group and US+MB+HGF-BMSCs group were significantly lowerthan the BMSCs group (P<0.05). On the time points of14d and21d, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were statistic significant difference (P<0.05). HGFin the renal tissue was detected by Real-time PCR on the four time points which suggestedthat on the time points of14d and28d, the US+MB+BMSCs group andUS+MB+HGF-BMSC group were significantly higher than the BMSCs group (P<0.05). However, the US+MB+HGF-BMSC group was significant different between groups after14d (P<0.05). Then theα-SMA was observed on the gene level and protein level (Westernblot detection, Immunohistochemical detection). The α-smooth muscle actin wasconsidered to be the sign of fibroblasts involved in interstitial fibrosis. The expression ofα-SMA mainly located in the interstitium and the difference was not significant on7d. After14d, the US+MB+BMSCs group and US+MB+HGF-BMSC group were significantly lowerthan the RUUO group and the BMSCs group (P<0.05).The expression of SDF-1was asfollow: The expression reached the peak on14d and the increasing levels in theUS+MB+BMSCs group and US+MB+HGF-BMSC group were significant higher than theRUUO group and the BMSCs group (P<0.05). With the time increased, the SDF-1expression gradually decreased. However, at the same time points, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were significant higher than the RUUO group andthe BMSCs group (P<0.05). At each time points, there were no significant differencebetween groups about the serum urea nitrogen, serum creatinine and glomerular filtrationrate (endogenous creatinine clearance Ccr).Conclusions:1. Successfully isolated, purified and cultured rat bone marrow derived BMSCs.2. Each experiment was arranged based on the uniform design. The optimaltransfection parameters of the acoustic intensity, the dosage of MBs, and the exposure timewere detected. Rat BMSCs were successfully transfected using MB mediated US combinedwith PEI. Iinitially identified this method does not change the BMSCs high proliferativecharacteristics and differentiation ability. The cell damage caused by UTMD canself-recovery and the fluorescence can continue expression.3. Preliminary study on the mechanism of BMSCs transfection induced by MBmediated US combined with PEI suggested one of the factors is cavitation, but thesynergistic effect may be the important reason.4. The healthy rat renal interstitial permeability was temporarily changed induced byMB mediated DUS which provids a new way for renal interstitial targeted delivery. Inaddition, preliminary study the mechanism of this method which suggested the cavitationmay be the major reason for the enhancement of renal interstitial capillary permeability.5. The rat UUO model and the rat RUUO model were successfully established which were confirmed by ultrasound imaging test, HE staining and Masson staining.6. MB mediated DUS can improve the exogenous BMSCs transplantation whichtargeted homing and aggregated to the renal interstitium of the UUO model.One of thepossible mechanism is the biological effects caused by UTMD can improve the BMSCssettlement, adhesion and migration towards the targeted area.The other possible mechanismis the paracrine action of the BMSCs. The paracrine action can improve SDF-1secretionand then tend more endogenous and exogenous BMSCs adhesion and homing to thetargeted renal tissue.7. The BMSCs transplantation and the HGF-BMSCs transplantation induced by MBmediated DUS can effectively delay the process of the development of renal interstitialfibrosis which significantly improved the damage kidney repairation comparing with thesimple BMSCs vein transplantation method.The cavatition erosion caused by MB mediatedDUS can change the renal interstitial micro-environment, improve the BMSCsconcentration in the target region and then delay the process of the development of renalinterstitial fibrosis and improve the renal tissue repairation.8. The HGF-BMSCs transplantation induced by MB mediated DUS can improve theHGF concentration in local area, inhibit the expression of the fibrogenic factor TGF-β,regulate and controlα-SMA, effectively inhibit renal interstitial collagen fiber formation,further promote the improvement of renal interstitial fibrosis.