Mesenchymal Stem Cells Ameliorate Peritoneal Fibrosis

Background: Peritoneal fibrosis can lead to serious clinical complications. Forexample, peritoneal dialysis (PD) for patients with end stage renal disease (ESRD) isseriously limited by ultrafiltration failure due to chronic peritoneal fibrosis, andpostoperative peritoneal adhesion due to acute peritoneal fibrosis is a major cause ofinfertility and bowel obstruction. Peritoneal mesothelial cells (PMCs) is important inthe maintenance of peritoneal homeostasis, including transport and movement of fluidand particulate material; the synthesis of pro-/anti-inflammatory cytokines, growthfactors, and extracellular matrix (ECM); and the control of fibrinolysis. Tissue repaircommences after acute or chronic mesothelial injury. This process is characterized byremesothelialization of the injured area and fibrosis with inflammations which lead toultrafiltration failure in PD or the formation of postoperative peritoneal adhesions. Inaddition, PMCs may undertake epithelial-myofibroblast transdifferentiation (EMT) toinitiate the process of fibrosis. Mesenchymal stem cells (MSCs) are efficient forrepairing injuries and reducing fibrosis. MSCs mediate their therapeutic effects byeither differentiating into functional reparative cells or by secreting paracrine factorsthat are mitogenic, cytoprotective, anti-inflammatory, anti-apoptotic, andanti-fibrogenic. To our limited knowledge, there has been no report about the effect ofMSCs on peritoneal fibrosis.Objective: This study was designed to investigate1.The effects of MSCs onmechanically injured PMCs and acute peritoneal fibrosis.2. The effects of MSCs onEMT of PMCs induced by PD and chronic peritoneal fibrosis. Besides, the underlyingmechanisms for MSCs to exert their therapeutic effects were to be investigated.Methods: This study is divided into two sections. One is the study of MSCsameliorate acute peritoneal fibrosis, the other is the study of MSCs ameliorate chronic peritoneal fibrosis.MSCs ameliorate acute peritoneal fibrosis: Sprague-Dawley (SD) rat acuteperitoneal adhesion model induced by mechanical scraping peritoneal injury wasestablished in vivo.5×106rat bone marrow-derived red fluorescent protein(RFP)-MSCs were injected into rats via the tail vein or peritoneum at different timesafter peritoneal scraping, the severity of peritoneal adhesions was evaluated bymacroscopic adhesion scoring at14d after scraping to determine the optimal injectionpathway and timing.5×106MSCs were injected into rats via the tail vein24h afterperitoneal scraping, the severity of peritoneal adhesions was evaluated bymacroscopic adhesion scoring after scraping, the inflammatory histological changeswere evaluated by hematoxylin-eosin staining (HE) staining, and the fibrogenichistological changes were evaluated by masson’s trichrome staining. The numbers ofneutrophils (myeloperoxidase (MPO)), macrophages (ED-1), fibroblasts (fibroblastspecific protein-1(FSP-1)) and the level of transforming growth factor β1(TGF-β1)were evaluated by immunohistochemical analysis. The number of PMCs (E-Cadherin)and the level of proliferating cell nuclear antigen (PCNA) were evaluated byimmunohistochemical analysis to evaluate the repair of PMCs. In vitro, primary ratperitoneal mesothelial cells (RPMCs) were isolated, cultured and identified.Mechanical scraped RPMCs were co-cultured with MSCs in Transwell system, themotility rates of cells entering the wound at different times were calculated,E-Cadherin and PCNA were evaluated by immunofluorescence analysis. MSCs waslabeled with near-infrared fluorescence lipophilic DiR, the dynamic distributions ofMSCs injected intravenously or intraperitoneally into rat or servere combinedimmune-deficiency (SCID) mouse at24h after peritoneal scraping were traced by invivo imaging. The distributions of MSCs in the lung, the spleen or other organs andthe orientation relationship with macrophages were detected by two-photon laserscanning fluorescence microscopy and immunofluorescence analysis. Theconcentrated serum-satrved conditioned medium (CM) of5×106MSCs was injectedinto rats via the tail vein24h,3d,5d after peritoneal scraping. The severity ofperitoneal adhesions was evaluated by macroscopic adhesion scoring after scraping,the fibrogenic histological changes were evaluated by masson’s trichrome staining. MSCs-CM (0h,12h,24h serum-starved respectively) were analyzed by antibodybased cytokine array to evaluate the secretions of cytokines of MSCs-CM. Cytokinewhich increased most dramatically was further verified by Enzyme-linkedimmunosorbent assay (ELISA). The expressions of tumor necrosis factor α(TNFα)-stimulating gene-6(TSG-6) in MSCs accumulated in the lung afterintravenous injection or in MSCs accumulated in the spleen after intraperitonealinjection were evaluated by immunofluorescence analysis, and the serumconcentrations of TSG-6were tested by ELISA. MSCs were transfected withTSG-6-small interfering (si) RNA to knockdown the expression and the secretion ofTSG-6in MSCs, the knockdown efficiency were evaluated by RT-PCR, Western blotand ELISA. In vivo, MSCs, or TSG-6-siRNA MSCs was injected into rats via the tailvein24h after peritoneal scraping, and MSCs-CM, TSG-6-siRNA MSCs-CM orrecombinant mouse TSG-6(rmTSG-6) was injected into rats via the tail vein24h,3d,5d after peritoneal scraping. The severity of peritoneal adhesions was evaluated bymacroscopic adhesion scoring after scraping, the fibrogenic histological changes wereevaluated by masson’s trichrome staining. In vitro, scraped RPMCs were co-culturedwith MSCs, TSG-6-siRNA MSCs or rmTSG-6in Transwell system, the motility ratesof cells entering the wound were calculated, E-Cadherin and PCNA were evaluatedby immunofluorescence analysis.MSCs ameliorate chronic peritoneal fibrosis: SD rat chronic peritoneal fibrosismodel induced by chronic peritoneal dialysis was established in vivo.5×106MSCswere injected into rats via the tail vein at different times after the initiation ofperitoneal dialysis fluid injection, the severity of peritoneal fibrosis was evaluated bymasson’s trichrome staining at28d after peritoneal dialysis to determine the optimalinjection timing.5×106MSCs were injected into rats via the tail vein immediatelyafter peritoneal dialysis, the ultrafiltration function and the transferration capacity ofglucose were evaluated at28d after peritoneal dialysis. The density of vessel inperitoneum was evaluated by HE staining, the fibrogenic histological changes wereevaluated by masson’s trichrome staining. The numbers of PMCs (E-Cadherin),myofibroblasts (smooth muscle actin α，α-SMA), and the level of TGF-β1wereevaluated by immunohistochemical analysis. Results: Injection of MSCs via the tail vein24h after peritoneal scraping reduced theperitoneal adhesion formation, while MSCs injected via peritoneum failed to reducethe adhesion. Reductions in adhesion formation; infiltration of neutrophils,macrophage cells; number of fibroblasts; and level of TGF-β1were found inMSCs-treated rat. In addition, the deposition of fibins and the formation of fibrousbands6d after scraping were also reduced. The proliferation and the repair ofmesothelial cells were stimulated by MSCs. In vitro, during the first2h after scraping,an increase in the migration of the RPMCs was observed when mechanical scrapedRPMCs were co-cultured in the Transwell with MSCs, the proliferation of injuredRPMCs co-cultured with MSCs increased3h after scraping; peak proliferationoccurred6h after scraping, which was6h ahead of the control group. MSCs injectedintravenously mainly accumulated in the lungs peaking at4h, the signals persisted forat least7d, MSCs injected intraperitoneally mainly accumulated in the spleen andliver with the peak at24h, the signals persisted for at least7d. But no apparent MSCswere observed either in the injured peritoneum, even in SCID mouse. Two-photonlaser scanning fluorescence microscopy and immunofluorescence analysisdemonstrated that emboli-like MSCs were trapped in the lungs4h after intravenousinjection, and the trapped MSC decreased dramatically within the first24h, but a fewMSCs still retained at7d with few swallowed by macrophages; While the majority ofMSCs accumulated in the spleen4h after intraperitoneal injection were swallowed bymacrophages, and the cell debris increased dramatically within24h. The injection ofserum-starved MSCs-CM intravenously reduced adhesions, similar to MSCs.Antibody based protein array of serum-free MSCs-CM showed that the releasing ofTNFα-stimulating gene (TSG)-6increased most dramatically. Within12h afterintravenous injection, MSCs accumulated in the lung could express TSG-6, and thelevels of TSG-6in serum were also increased. While MSCs accumulated in the spleenafter intraperitoneal injection did not express TSG-6, and the levels of TSG-6inserum were not different from those in the control group. Knocked down of TSG-6inexpression and secretion, MSCs trapped in the lung could not express TSG-6, andTSG-6in serum was not increased. In addition, promotion of mesothelial cell repairand reduction of peritoneal adhesion were produced by the administration of rmTSG-6, and were weakened by TSG-6-RNA interfering.Injection of MSCs via the tail vein immediately after peritoneal dialysis reducedthe peritoneal fibrosis. The ultrafiltration function and the transferration capacity ofglucose in MSCs treated group were similar to that in the normal group. Whilecompared with the control group, the density of peritoneal vessel in MSCs treatedgroup was much lower, and the severity of peritoneal fibrosis was reduced. Thenumber of PMCs in peritoneum was increased at7d after peritoneal dialysis, but thenumber of myofibroblasts at21d,28d, and the level of TGF-β1were decreased.Conclusion: These results indicate that intravenously injected MSCs may promoteperitoneal injury by reducing the inflitrations of inflammatory cells, the numbers offibroblats, the level of TGF-β1, as well as by stimulating the proliferation andmigration of mesothelial cells. Rather than the engraftment, the distant action of thesecretion of TSG-6into the blood by MSCs accumulated in the lung makes a majorcontribution to the therapeutic benefits of MSCs. In addition, MSCs could inhibitEMT of PMCs induced by peritoneal dialysis fluid, hence ameliorate peritonealfibrosis and improve the ultrafiltration function of peritoneum.