The Fibronectin EIIIA Splice Variant Promotes Hepatic Stellate Cell Motility and Liver Fibrosis via α\u3csub\u3e9\u3c/sub\u3eβ\u3csub\u3e1\u3c/sub\u3e Integrin

Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA+ cFN) is upregulated after liver injury and has been reported to promote myofibroblast differentiation of precursor cells from liver, lung, and skin. We examined the role of EIIIA+ cFN in liver fibrosis induced by thioacetamide toxicity and by bile duct ligation. Surprisingly, EIIIA -/- and wild type mice were equally susceptible to fibrosis after bile duct ligation, but not after thioacetamide. We therefore studied the effects of EIIIA+ cFN on two major myofibroblast precursor populations in the liver, hepatic stellate cells and portal fibroblasts, which we suggest are the dominant cellular mediators of hepatic versus biliary fibrosis, respectively. Using a mechanically physiologic cell culture system, we found that EIIIA+ cFN was not required for myofibroblast differentiation of either cell type, but had cell-specific effects on motility. Hepatic stellate cells cultured on cFN were more motile than cells on plasma fibronectin (pFN), which lacks EIIIA, and this increased motility was dependent on EIIIA and its specific receptor, integrin α 9 β1. Portal fibroblasts, in contrast, expressed little α9 β1 and demonstrated no motility increase on EIIIA+ cFN compared to pFN. These data demonstrate that myofibroblast populations have significant functional differences regulated by subtle changes in the matrix environment, and they suggest that variable responses to matrix proteins may drive different forms of fibrosis

The fibronectin EIIIA splice variant promotes hepatic stellate cell motility and liver fibrosis via alpha9beta1 integrin

Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA+ cFN) is upregulated after liver injury and has been reported to promote myofibroblast differentiation of precursor cells from liver, lung, and skin. We examined the role of EIIIA+ cFN in liver fibrosis induced by thioacetamide toxicity and by bile duct ligation. Surprisingly, EIIIA-/- and wild type mice were equally susceptible to fibrosis after bile duct ligation, but not after thioacetamide. We therefore studied the effects of EIIIA+ cFN on two major myofibroblast precursor populations in the liver, hepatic stellate cells and portal fibroblasts, which we suggest are the dominant cellular mediators of hepatic versus biliary fibrosis, respectively. Using a mechanically physiologic cell culture system, we found that EIIIA+ cFN was not required for myofibroblast differentiation of either cell type, but had cell-specific effects on motility. Hepatic stellate cells cultured on cFN were more motile than cells on plasma fibronectin (pFN), which lacks EIIIA, and this increased motility was dependent on EIIIA and its specific receptor, integrin α9β1. Portal fibroblasts, in contrast, expressed little α9β1 and demonstrated no motility increase on EIIIA+ cFN compared to pFN. These data demonstrate that myofibroblast populations have significant functional differences regulated by subtle changes in the matrix environment, and they suggest that variable responses to matrix proteins may drive different forms of fibrosis

The fibronectin EIIIA splice variant promotes hepatic stellate cell motility and liver fibrosis via alpha9beta1 integrin

Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA+ cFN) is upregulated after liver injury and has been reported to promote myofibroblast differentiation of precursor cells from liver, lung, and skin. We examined the role of EIIIA+ cFN in liver fibrosis induced by thioacetamide toxicity and by bile duct ligation. Surprisingly, EIIIA-/- and wild type mice were equally susceptible to fibrosis after bile duct ligation, but not after thioacetamide. We therefore studied the effects of EIIIA+ cFN on two major myofibroblast precursor populations in the liver, hepatic stellate cells and portal fibroblasts, which we suggest are the dominant cellular mediators of hepatic versus biliary fibrosis, respectively. Using a mechanically physiologic cell culture system, we found that EIIIA+ cFN was not required for myofibroblast differentiation of either cell type, but had cell-specific effects on motility. Hepatic stellate cells cultured on cFN were more motile than cells on plasma fibronectin (pFN), which lacks EIIIA, and this increased motility was dependent on EIIIA and its specific receptor, integrin α9β1. Portal fibroblasts, in contrast, expressed little α9β1 and demonstrated no motility increase on EIIIA+ cFN compared to pFN. These data demonstrate that myofibroblast populations have significant functional differences regulated by subtle changes in the matrix environment, and they suggest that variable responses to matrix proteins may drive different forms of fibrosis

Designer blood: creating hematopoietic lineages from embryonic stem cells

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases

α-synuclein impairs autophagosome maturation through abnormal actin stabilization.

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson's disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson's disease and related α-synucleinopathies

Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy

© 2020, The Author(s). The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson’s disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets we performed proteomic analysis in a highly penetrant new Drosophila model of α-synucleinopathy. We identified 476 significantly upregulated and 563 significantly downregulated proteins in heads from α-synucleinopathy model flies compared to controls. We then used multiple complementary analyses to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. We performed Gene Ontology enrichment analysis, integrated our proteomic changes with human Parkinson’s disease genetic studies, and compared the α-synucleinopathy proteome with that of tauopathy model flies, which are relevant to Alzheimer’s disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, we found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. Our integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. Here we outline and implement an integrative approach to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila

Downregulation of the tyrosine degradation pathway extends Drosophila lifespan

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.publishe

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN