Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis.

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-ß1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Evidence for discrete modes of YAP1 signaling via mRNA splice isoforms in development and diseases.

Yes-associated protein 1 (YAP1) is a transcriptional co-activator downstream of Hippo pathway. The pathway exerts crucial roles in organogenesis and its dysregulation is associated with the spreading of different cancer types. YAP1 gene encodes for multiple protein isoforms, whose specific functions are not well defined. We demonstrate the splicing of isoform-specific mRNAs is controlled in a stage- and tissue-specific fashion. We designed expression vectors encoding for the most-represented isoforms of YAP1 with either one or two WW domains and studied their specific signaling activities in YAP1 knock-out cell lines. YAP1 isoforms display both common and unique functions and activate distinct transcriptional programs, as the result of their unique protein interactomes. By generating TEAD-based transcriptional reporter cell lines, we demonstrate individual YAP1 isoforms display unique effects on cell proliferation and differentiation. Finally, we illustrate the complexity of the regulation of Hippo-YAP1 effector in physiological and in pathological conditions of the heart.

Ultrastructural and cytochemical changes in the liver of primary biliary cirrhosis patients.

The aim of this paper is to establish whether there are cytochemical or ultrastructural alterations in the hepatocytes of patients with primary biliary cirrhosis (PBC) at stages I and II compared with the biopsies from individuals with normal liver. Cytochemical technique with ATP as substrate, transmission electron microscopy (TEM) and freeze fracture were used for the studies. In the normal liver biopsies the ultrastructural cytochemical localization of the enzymatic activity was clearly shown in the bile canaliculi. In the PBC biopsies, the enzymatic activity is increased in the bile canaliculi and is also present in the lateral membranes of the hepatocyte. TEM of the lateral surface of the hepatocyte in normal livers showed a smooth surface without microvilli but in PBC livers a large number of microvilli were seen in the lateral membranes. The Golgi apparatus in these patients was localized not only near the canaliculi (normal livers) but also in front of the microvilli. Freeze-fracture showed normal features in the bile canaliculus junctions of the PBC patients. We suggest that the localization of the enzymatic reaction, microvilli and Golgi apparatus at the PBC hepatocyte lateral membranes may represent a compensatory mechanism for derivation of bile flow and other components from the hepatocyte to the intercellular space.

Ultrastructural and cytochemical changes in the liver of primary biliary cirrhosis patients

The aim of this paper is to establish whether there are cytochemical or ultrastructural alterations in the hepatocytes of patients with primary biliary cirrhosis (PBC) at stages I and II compared with the biopsies from individuals with normal liver. Cytochemical technique with ATP as substrate, transmission electron microscopy (TEM) and freeze fracture were used for the studies. In the normal liver biopsies the ultrastructural cytochemical localization of the enzymatic activity was clearly shown in the bile canaliculi. In the PBC biopsies, the enzymatic activity is increased in the bile canaliculi and is also present in the lateral membranes of the hepatocyte. TEM of the lateral surface of the hepatocyte in normal livers showed a smooth surface without microvilli but in PBC livers a large number of microvilli were seen in the lateral membranes. The Golgi apparatus in these patients was localized not only near the canaliculi (normal livers) but also in front of the microvilli. Freeze-fracture showed normal features in the bile canaliculus junctions of the PBC patients. We suggest that the localization of the enzymatic reaction, microvilli and Golgi apparatus at the PBC hepatocyte lateral membranes may represent a compensatory mechanism for derivation of bile flow and other components from the hepatocyte to the intercellular space.

Ultrastructural and cytochemical changes in the liver of primary biliary cirrhosis patients

The aim of this paper is to establish whether there are cytochemical or ultrastructural alterations in the hepatocytes of patients with primary biliary cirrhosis (PBC) at stages I and II compared with the biopsies from individuals with normal liver. Cytochemical technique with ATP as substrate, transmission electron microscopy (TEM) and freeze fracture were used for the studies. In the normal liver biopsies the ultrastructural cytochemical localization of the enzymatic activity was clearly shown in the bile canaliculi. In the PBC biopsies, the enzymatic activity is increased in the bile canaliculi and is also present in the lateral membranes of the hepatocyte. TEM of the lateral surface of the hepatocyte in normal livers showed a smooth surface without microvilli but in PBC livers a large number of microvilli were seen in the lateral membranes. The Golgi apparatus in these patients was localized not only near the canaliculi (normal livers) but also in front of the microvilli. Freeze-fracture showed normal features in the bile canaliculus junctions of the PBC patients. We suggest that the localization of the enzymatic reaction, microvilli and Golgi apparatus at the PBC hepatocyte lateral membranes may represent a compensatory mechanism for derivation of bile flow and other components from the hepatocyte to the intercellular space.(AU)

Ultrastructural and cytochemical changes in the liver of primary biliary cirrhosis patients.

The aim of this paper is to establish whether there are cytochemical or ultrastructural alterations in the hepatocytes of patients with primary biliary cirrhosis (PBC) at stages I and II compared with the biopsies from individuals with normal liver. Cytochemical technique with ATP as substrate, transmission electron microscopy (TEM) and freeze fracture were used for the studies. In the normal liver biopsies the ultrastructural cytochemical localization of the enzymatic activity was clearly shown in the bile canaliculi. In the PBC biopsies, the enzymatic activity is increased in the bile canaliculi and is also present in the lateral membranes of the hepatocyte. TEM of the lateral surface of the hepatocyte in normal livers showed a smooth surface without microvilli but in PBC livers a large number of microvilli were seen in the lateral membranes. The Golgi apparatus in these patients was localized not only near the canaliculi (normal livers) but also in front of the microvilli. Freeze-fracture showed normal features in the bile canaliculus junctions of the PBC patients. We suggest that the localization of the enzymatic reaction, microvilli and Golgi apparatus at the PBC hepatocyte lateral membranes may represent a compensatory mechanism for derivation of bile flow and other components from the hepatocyte to the intercellular space.