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Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice.

ABSTRACT: Transplantation of healthy cells to repair organ damage or replace deficient functions constitutes a major goal of cell therapy. However, the mechanisms by which transplanted cells engraft, proliferate, and function remain unknown. To investigate whether host liver sinusoidal endothelium could be replaced with transplanted liver sinusoidal endothelial cells, we developed an animal model of tissue replacement that utilized a genetic system to identify transplanted cells and induced host-cell perturbations to confer a proliferative advantage to transplanted cells. Under these experimental conditions, transplanted cells engrafted efficiently and proliferated to replace substantial portions of the liver endothelium. Tissue studies demonstrated that transplanted cells became integral to the liver structure and reacquired characteristic endothelial morphology. Characterization of transplanted endothelial cells by membrane markers and studies of cellular function, including synthesis and release of coagulation factor VIII, demonstrated that transplanted cells were functionally intact. Further analysis showed that repopulation of the livers of mice that model hemophilia A with healthy endothelial cells restored plasma factor VIII activity and corrected their bleeding phenotype. Our studies therefore suggest that transplantation of healthy endothelial cells should be considered for cell therapy of relevant disorders and that endothelial reconstitution with transplanted cells may offer an excellent paradigm for defining organ-specific pathophysiological mechanisms.

SUBMITTER: Follenzi A

PROVIDER: S-EPMC2242617 | biostudies-literature |

REPOSITORIES: biostudies-literature

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GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice

Project description:Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic FVIII amounts and correction of hemophilia A mice.

| S-SCDT-10_1038-S44321-024-00072-8 | biostudies-other

Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice.

Project description:Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

| S-EPMC9631574 | biostudies-literature

Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

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| S-EPMC3805584 | biostudies-literature

Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells.

Project description:Our previous studies have demonstrated that platelet FVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice, even in the presence of inhibitory antibodies, but none of our studies has targeted human cells. Here, we evaluated the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A. Human platelet FVIII expression was introduced by 2bF8LV-mediated transduction of human cord blood (hCB) CD34(+) cells followed by xenotransplantation into immunocompromised NSG mice or NSG mice in an FVIII(null) background (NSGF8KO). Platelet FVIII was detected in all recipients that received 2bF8LV-transduced hCB cells as long as human platelet chimerism persisted. All NSGF8KO recipients (n = 7) that received 2bF8LV-transduced hCB cells survived tail clipping if animals had greater than 2% of platelets derived from 2bF8LV-transduced hCB cells, whereas 5 of 7 survived when human platelets were 0.3% to 2%. Whole blood clotting time analysis confirmed that hemostasis was improved in NSGF8KO mice that received 2bF8LV-transduced hCB cells. We demonstrate, for the first time, the feasibility of 2bF8LV gene delivery to human hematopoietic stem cells to introduce FVIII expression in human platelets and that human platelet-derived FVIII can improve hemostasis in hemophilia A.

| S-EPMC3894495 | biostudies-literature

Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype.

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| S-EPMC4697439 | biostudies-literature

Fetal liver cells transplanted in utero rescue the osteopetrotic phenotype in the oc/oc mouse.

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| S-EPMC2665735 | biostudies-literature

Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes.

Project description:Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes.Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL.Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itg?4, HNF1?, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes.Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.

| S-EPMC5137249 | biostudies-literature

LSEC Fenestrae Are Preserved Despite Pro-inflammatory Phenotype of Liver Sinusoidal Endothelial Cells in Mice on High Fat Diet.

Project description:Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2-8 week) and late (15-20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE2 and PGF2?) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.

| S-EPMC6379824 | biostudies-literature

Implantation of healthy matrix-embedded endothelial cells rescues dysfunctional endothelium and ischaemic tissue in liver engraftment.

Project description:ObjectiveLiver transplantation is limited by ischaemic injury which promotes endothelial cell and hepatocyte dysfunction and eventually organ failure. We sought to understand how endothelial state determines liver recovery after hepatectomy and engraftment.DesignMatrix-embedded endothelial cells (MEECs) with retained healthy phenotype or control acellular matrices were implanted in direct contact with the remaining median lobe of donor mice undergoing partial hepatectomy (70%), or in the interface between the remaining median lobe and an autograft or isograft from the left lobe in hepatectomised recipient mice. Hepatic vascular architecture, DNA fragmentation and apoptosis in the median lobe and grafts, serum markers of liver damage and phenotype of macrophage and lymphocyte subsets in the liver after engraftment were analysed 7 days post-op.ResultsHealthy MEECs create a functional vascular splice in donor and recipient liver after 70% hepatectomy in mouse protecting these livers from ischaemic injury, hepatic congestion and inflammation. Macrophages recruited adjacent to the vascular nodes into the implants switched to an anti-inflammatory and regenerative profile M2. MEECs improved liver function and the rate of liver regeneration and prevented apoptosis in donor liver lobes, autologous grafts and syngeneic engraftment.ConclusionsImplants with healthy endothelial cells rescue liver donor and recipient endothelium and parenchyma from ischaemic injury after major hepatectomy and engraftment. This study highlights endothelial-hepatocyte crosstalk in hepatic repair and provides a promising new approach to improve regenerative medicine outcomes and liver transplantation.

| S-EPMC5288307 | biostudies-literature

Spermidine Supplementation Protects the Liver Endothelium from Liver Damage in Mice.

Project description:Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl4 and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl4 and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression.

| S-EPMC8617984 | biostudies-literature

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