De novo formation of the biliary system by TGFß-mediated hepatocyte transdifferentiation.

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development-the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3-6. In contrast to bile duct development7-9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFß signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFß signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.

Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration.

Hepatocyte proliferation is the principal mechanism for generating new hepatocytes in liver homeostasis and regeneration. Recent studies have suggested that this ability is not equally distributed among hepatocytes but concentrated in a small subset of hepatocytes acting like stem cells, located around the central vein or distributed throughout the liver lobule and exhibiting active WNT signaling or high telomerase activity, respectively. These findings were obtained by utilizing components of these growth regulators as markers for genetic lineage tracing. Here, we used random lineage tracing to localize and quantify clonal expansion of hepatocytes in normal and injured liver. We found that modest proliferation of hepatocytes distributed throughout the lobule maintains the hepatocyte mass and that most hepatocytes proliferate to regenerate it, with diploidy providing a growth advantage over polyploidy. These results show that the ability to proliferate is broadly distributed among hepatocytes rather than limited to a rare stem cell-like population.

In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis.

Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.

Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2.

Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.

A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.