Vitamin A-containing Lipid Droplets Research Articles

Fusion Protein of RBP and Albumin Domain III Reduces Lung Fibrosis by Inactivating Lung Stellate Cells.

Activated stellate cells play a role in fibrosis development in the liver, pancreas, and kidneys. The fusion protein R-III, which consists of retinol-binding protein and albumin domain III, has been demonstrated to attenuate liver and renal fibrosis by suppressing stellate cell activation. In this study, we investigated the efficacy of R-III against bleomycin-induced lung fibrosis in mice. R-III reduced lung fibrosis and primarily localized in autofluorescent cells in the lung tissue. Furthermore, we isolated lung stellate cells (LSCs) from rat lungs using the isolation protocol employed for hepatic stellate cells (HSCs). LSCs shared many characteristics with HSCs, including the presence of vitamin A-containing lipid droplets and the expression of alpha-smooth muscle actin and collagen type I, markers for activated HSCs/myofibroblasts. LSCs spontaneously transdifferentiated into myofibroblasts in in vitro culture, which was inhibited by R-III. These findings suggest that R-III may reduce lung fibrosis by inactivating LSCs and could be a promising treatment for extrahepatic fibrosis.

Inhibition of Renal Stellate Cell Activation Reduces Renal Fibrosis

Interstitial fibrosis is a common feature of chronic kidney disease, and platelet-derived growth factor receptor-β (PDGFR-β)-positive mesenchymal cells are reportedly the major source of scar-producing myofibroblasts. We had previously demonstrated that albumin and its derivative R-III (a retinol-binding protein-albumin domain III fusion protein) inhibited the transdifferentiation/activation of hepatic stellate cells (HSCs) to myofibroblasts and that R-III administration reduced liver fibrosis. In this study, we isolated cells (referred to as renal stellate cells, RSCs) from rat kidney tissues using the HSC isolation protocol and compared their morphological and biochemical characteristics with those of HSCs. RSCs shared many characteristics with HSCs, such as storage of vitamin A-containing lipid droplets and expression of HSC markers as well as pericyte markers. RSCs underwent spontaneous transdifferentiation into myofibroblasts in in vitro culture, which was inhibited by albumin expression or R-III treatment. We also evaluated the therapeutic effects of R-III in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Injected R-III localized predominantly in cytoglobin/stellate cell activation-associated protein (Cygb/STAP)-positive cells in the kidney and reduced renal fibrosis. These findings suggest that RSCs can be recognized as the renal counterparts of HSCs and that RSCs represent an attractive therapeutic target for anti-fibrotic therapy.

Metabolic Signature of Hepatic Fibrosis: From Individual Pathways to Systems Biology.

Hepatic fibrosis is a major cause of morbidity and mortality worldwide, as it ultimately leads to cirrhosis, which is estimated to affect up to 2% of the global population. Hepatic fibrosis is confirmed by liver biopsy, and the erroneous nature of this technique necessitates the search for noninvasive alternatives. However, current biomarker algorithms for hepatic fibrosis have many limitations. Given that the liver is the largest organ and a major metabolic hub in the body, probing the metabolic signature of hepatic fibrosis holds promise for the discovery of new markers and therapeutic targets. Regarding individual metabolic pathways, accumulating evidence shows that hepatic fibrosis leads to alterations in carbohydrate metabolism, as aerobic glycolysis is aggravated in activated hepatic stellate cells (HSCs) and the whole fibrotic liver; in amino acid metabolism, as Fischer’s ratio (branched-chain amino acids/aromatic amino acids) decreases in patients with hepatic fibrosis; and in lipid metabolism, as HSCs lose vitamin A-containing lipid droplets during transdifferentiation, and cirrhotic patients have decreased serum lipids. The current review also summarizes recent findings of metabolic alterations relevant to hepatic fibrosis based on systems biology approaches, including transcriptomics, proteomics, and metabolomics in vitro, in animal models and in humans.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

Hepatic stellate cell – vitamin A-rich cells

Quiescent hepatic stellate cells (HSCs) are characterized by their localization of perisinusoidal cells and their long cytoplasmic processes, extending both along and around the sinusoids as well as between the hepatocytes. These long cytoplasmic processes, which have numerous vitamin A-containing lipid droplets, are essential for the identification of HSCs in the quiescent phase. Glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, UK) is another crucial marker for HSCs which may allow a distinction to be made between the HSCs and other fibroblastic liver cells. However, induction of alpha-smooth muscle actin (α-SMA) is the most reliable marker of stellate cell activation because it is absent from other resident hepatocytes in a normal or injured liver, with the exception of the smooth muscle cells surrounding large vessels where it is present. Desmin has been used as a typical marker for contractile stellate cells in rodent livers; however, its expression is unreliable in humans. Furthermore, HSCs' transdifferentiation to the active phase has been proven to rely on E-cadherin switching to N-cadherin during HSC activation. Significant evidence now exists to consider HSCs as the main matrix-producing cells in the process of the liver fibrosis. Liver injury, regardless of the aetiology, tumour growth and metastases will ultimately lead to activation of HSCs. Several studies have dissected the molecular mechanisms involved in liver fibrosis, most of which are intimately connected to HSCs. As a result, a number of key steps in the process of stellate cell activation and fibrogenesis have been identified as potential therapeutic targets that may be clinically useful in preventing, or treating, liver fibrosis.

Retinol binding protein-albumin domain III fusion protein deactivates hepatic stellate cells.

Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis.

Loss of vitamin A-containing lipid droplets in hepatic stellate cells and liver fibrosis

Loss of vitamin A-containing lipid droplets in hepatic stellate cells and liver fibrosis

Expression of the Peroxisome Proliferator-Activated Receptors (PPARs) in the Hepatic Stellate Cells

Hepatic stellate cells (HSCs) have an important role in maintaining vitamin A (retinoid) homeostasis [1,2]. Peroxisome proliferator-activated receptors (PPARs) are members of the steroid/retinoid nuclear hormone receptor superfamily of ligand-activated transcription factors, form a heterodimer with retinoid X receptor (RXR), and play an important role in lipid metabolisms. Several reports [3-5] have suggested that expression of PPAR-gamma was reduced with the acquisition of activated phenotype (termed as activation) such as lack of cytoplasmic lipid droplets containing vitamin A in subcultured HSC. Previously, we showed that the subcultured HSCs restored the cytoplasmic lipid droplets emanating vitamin A autofluorescence by the addition of retinyl acetate to culture medium [6]. However, it remains unclear whether or not PPAR-gamma expression is involved in formation of vitamin A-containing lipid droplets and maintaining of vitamin A homeostasis in HSCs. In this study, we examined roles of PPARs, particularly PPAR-gamma, in accumulation of vitamin A-containing lipid droplets in cultivated HSCs.

INDUCTION OF CELLULAR PROCESSES CONTAINING COLLAGENASE AND RETINOID BY INTEGRIN-BINDING TO INTERSTITIAL COLLAGEN IN HEPATIC STELLATE CELL CULTURE

Cultered hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti-integrin α2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets. The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.

Detection of retinyl palmitate and retinol in the liver of mice injected with excessive amounts of retinyl acetate.

The transport of subcutaneously injected retinyl acetate (RA, 100,000 IU/mouse, 105,470 nM) was investigated in male ICR mice (10-week-old) at 0, 3, 6, 12, 18, 24 and 72 hr after a single injection. The retinol and retinyl palmitate levels of liver homogenates, bile in the gallbladder and serum from peripheral blood were measured by high performance liquid chromatography (HPLC) method. Retinyl palmitate in the lipid droplets of hepatocytes and Ito cells was localized by a modified gold chloride staining method. Accumulation of retinyl palmitate peaked at 12 hr post-injection and decreased thereafter until 24 hr post-injection. Fluorescence microscopy revealed many fluorescent vitamin A-containing lipid droplets in hepatocytes around central veins at 12 hr post-injection, but such droplets were not observed in the vehicle control mice or at in the RA-injected mice after 18 hr of injection. Electron microscopic observation also indicated that many retinyl esters-containing lipid droplets were observed in hepatocytes around the central veins at 12 hr post-injection, but no droplets were seen in the controls or 18 hr post-injection. The retinyl palmitate levels in liver homogenates assessed by HPLC decreased from 12 to 24 hr post-injection and increased significantly in bile, while retinol in liver homogenates and serum markedly increased. One of the morphological alterations was intense vacuolization in hepatocyte cell cords from the portal toward the central vein observed at 24 hr post-injection. Transitional lipid droplets between vacuoles and lipid droplets were identified in those hepatocytes. These results of HPLC analysis of retinol and retinyl palmitate in liver homogenates, serum, and bile, together with the results of gold chloride staining suggested that subcutaneously injected RA was first incorporated in hepatocytes at 12 hr and then partially metabolized through vacuoles, transferred into the blood and secreted into the bile over a 24 hr period. Many retinyl esters-containing lipid droplets were visualized in Ito cells at 72 hr post-injection. Most of vitamin A in the liver homogenates measured by HPLC was retinyl palmitate. Therefore, the contents in those lipid droplets might be retinyl palmitate.

The morphology of release of vitamin A‐containing lipid droplets by hepatocytes in rat liver

Vitamin A-containing lipid droplets in the hepatocytes of rat liver were found to be exocytotically released from the cells in the form of a "lipid droplet--retinol-binding protein (RBP)--immunoreactive complex" following intraportal injection of retinol (17, 33, 67, or 100 micrograms). Evidence that the lipid droplets contain vitamin A was obtained by fluorescence microscopy of vitamin A. Intraportal injection of retinol produced varied numbers and sizes of vacuoles in the hepatocytes. The substance within the vacuoles exhibited a meshwork-like configuration in sections from slices incubated in a medium for revealing acid phosphatase activity or the corresponding control medium and was RBP-immunoreactive and proteinaceous in nature. The occurrence and number of the vacuoles depended on the dosage of injected retinol, being greatest at a dosage of 100 micrograms of retinol and becoming progressively less at dosages of 67, 33, and 17 micrograms. The vacuoles were formed by vacuolization of cisternae of the rough endoplasmic reticulum. The formation of vacuoles reached a maximum 30 min after intraportal injection of 100 micrograms retinol, and the vacuoles and lipid droplets had almost disappeared from the hepatocytes after 90 min. Little or no esterase activity was found in lipid droplets in the hepatocytes before intraportal injection of retinol, but after the injection, lipid droplets that had fused with the vacuoles become strongly positive for this enzyme activity. This suggests that hydrolysis of retinyl esters may occur in the process of complex formation in rat hepatocytes.

Morphology of the release of vitamin A-containing lipid droplets by perisinusoidal lipocytes in the rat liver.

Vitamin A-containing lipid droplets in perisinsoidal lipocytes (or fat-storing cells) of rat liver were found to be exocytotically released from the cells in the form of a 'lipid droplet-protein complex' following intraportal injection of retinol (17, 33, 67 or 100 micrograms). Intraportal injection of retinol produced varied numbers and sizes of vacuoles with a limiting membrane in perisinusoidal lipocytes. The substance within the vacuoles exhibited a meshwork-like configuration in sections from slices incubated in a medium for revealing acid phosphatase activity or the corresponding control medium, and was immunoreactive to retinol-binding protein and proteinaceous in nature. The occurrence and number of the vacuoles depended on the dosage of injected retinol, being greatest at a dosage of 100 micrograms of retinol and becoming progressively less at dosages of 67, 33 and 17 micrograms. The vacuoles appeared to be formed by vacuolization of cisternae of the rough endoplasmic reticulum. Little or no esterase activity was found in lipid droplets in perisinusoidal lipocytes before the intraportal injection of retinol, but after the injection lipid droplets which had fused with the vacuoles became strongly positive for this enzyme activity. This suggests that hydrolysis of retinyl esters may occur in the process of complex formation in perisinusoidal lipocytes.

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver.

Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane. To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups. No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.

Localization of tritiated vitamin A in lymph nodes of the mouse: an autoradiographic study of vitamin A-storing cells.

Localization of tritiated vitamin A in lymph nodes of the mouse was investigated by the use of light- and electron-microscopic autoradiography. Young male mice were fed a diet containing 3H-vitamin A acetate for a week. Lymph nodes were removed and prepared for autoradiography. Lipid droplets in fibroblast-like cells showed high concentrations of radioactivity. These cells were distributed around lymphatic sinuses and blood vessels. The cells can, therefore, be classified as "vitamin A-storing cells" according to criteria proposed earlier by Yamada and Hirosawa (1976). Control animals from the same litter were maintained on ordinary laboratory food for the same period and examined by electron microscopy. Lipid-droplet-containing cells were found in the same areas as in the experimental animals but in fewer numbers. This suggests that the increased number of vitamin A-containing lipid droplets is due to hypervitaminosis A in the experimental animals. The presence of some cells containing these droplets in the control animals would imply that even under normal feeding conditions the animals ingested excess amounts of vitamin A, which was retained in lipid droplets. The stored vitamin A probably participates in metabolic processes such as the formation of glycoproteins in ground substance.