single-particle-interferometric-reflectance-imaging-characterization

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

University of Kansas Medical Center

Cell-to-cell communication is a fascinating process that is essential for maintaining tissue and whole-body homeostasis. Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles that are a means of communication between cells. Accumulating evidence indicates that EVs can render either beneficial or harmful outcomes, depending on the specific cargos (e.g. proteins, lipids, RNAs) transferred between cells. EVs also have great value as diagnostic and prognostic markers of disease because they are present in a variety of biological fluids and carry bioactive molecules from their cells or tissues of origin. Liver cells can both release and receive EVs derived from other cells and emerging evidence indicates that liver EVs play important roles in the pathogenesis of various liver diseases, including liver cancer, viral hepatitis, non-alcoholic fatty liver disease, and alcoholic liver disease. This review provides an overview of the biogenesis and secretion of EVs and summarizes the most recent advances in understanding the role of EVs in liver physiology and diseases. Additionally, we discuss potential applications of liver EVs as biomarkers and in therapeutic approaches to treat liver diseases.

Decoding the Role of Extracellular Vesicles in Liver Diseases.

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, ) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide , we demonstrated that the suppression of  expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun-mediated transcriptional repression of  Interestingly,  induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C-C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH.

Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis.

Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation.

Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers.

Autophagy is a lysosomal degradation pathway that degrades cytoplasmic proteins and organelles. Absence of autophagy in hepatocytes has been linked to promoting liver injury and tumorigenesis; however, the mechanisms behind why a lack of autophagy induces these complications are not fully understood. The role of mammalian target of rapamycin (mTOR) in impaired autophagy-induced liver pathogenesis and tumorigenesis was investigated by using liver-specific autophagy related 5 knockout (L-ATG5 KO) mice, L-ATG5/mTOR, and L-ATG5/Raptor double knockout (DKO) mice. We found that deletion of mTOR or Raptor in L-ATG5 KO mice at 2 months of age attenuated hepatomegaly, cell death, and inflammation but not fibrosis. Surprisingly, at 6 months of age, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice also had increased hepatic inflammation, fibrosis, and liver injury, similar to the L-ATG5 KO mice. Moreover, more than 50% of L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice already developed spontaneous tumors, but none of the L-ATG5 KO mice had developed any tumors at 6 months of age. At 9 months of age, all L-ATG5/mTOR DKO and L-ATG5/Raptor DKO had developed liver tumors. Mechanistically, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice had decreased levels of hepatic ubiquitinated proteins and persistent nuclear erythroid 2 p45-related factor 2 activation but had increased Akt activation compared with L-ATG5 KO mice. Conclusion: Loss of mTOR signaling attenuates the liver pathogenesis in mice with impaired hepatic autophagy but paradoxically promotes tumorigenesis in mice at a relatively young age. Therefore, the balance of mTOR is critical in regulating the liver pathogenesis and tumorigenesis in mice with impaired hepatic autophagy.

Dual Roles of Mammalian Target of Rapamycin in Regulating Liver Injury and Tumorigenesis in Autophagy-Defective Mouse Liver.

Exosomes are small membrane vesicles (ranging from 30 nm to 150 nm), secreted by different cell types upon fusion of multivesicular bodies (MVB) to the cell plasma membrane under a variety of normal and pathological conditions. Through transferring their cargos such as proteins, lipids and nucleic acids from donor cells to recipient cells, exosomes play a crucial role in cell-to-cell communication. Due to their presence in most body fluids (such as blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal and peritoneal fluids), and their role in carrying bioactive molecules from the cells of origin, exosomes have attracted great interest in their diagnostic and prognostic value for various diseases and therapeutic approaches. Although a large body of literature has documented the importance of exosomes over the past decade, there is no article systematically summarizing protein markers of exosome from different resources and the antibodies that are suited to characterize exosomes. In this review, we briefly summarize the exosome marker proteins, exosomal biomarkers for different diseases, and the antibodies suitable for different bio-resources exosomes characterization.

A review on protein markers of exosome from different bio-resources and the antibodies used for characterization.

Alzheimer's disease (AD) is the most common neurodegenerative disease, yet there are no disease-modifying treatments available and there is no cure. It is becoming apparent that metabolic and vascular conditions such as type 2 diabetes (T2D) and hypertension promote the development and accumulation of Alzheimer's disease-related dementia pathologies. To this end, aerobic exercise, which is a common lifestyle intervention for both metabolic disease and hypertension, is shown to improve brain health during both healthy aging and dementia. However, noncompliance or other barriers to exercise response are common in exercise treatment paradigms. In addition, reduced intracellular proteostasis and mitochondrial function could contribute to the etiology of AD. Specifically, compromised chaperone systems [i.e., heat shock protein (HSP) systems] can contribute to protein aggregates (i.e., β-amyloid plaques and neurofibrillary tangles) and reduced mitochondrial quality control (i.e., mitophagy). Therefore, novel therapies that target whole body metabolism, the vasculature, and chaperone systems (like HSPs) are needed to effectively treat AD. This review focuses on the role of heat therapy in the treatment and prevention of AD. Heat therapy has been independently shown to reduce whole body insulin resistance, improve vascular function, activate interorgan cross talk via endocytic vesicles, and activate HSPs to improve mitochondrial function and proteostasis in a variety of tissues. Thus, heat therapy could offer immense clinical benefit to patients suffering from AD. Importantly, future studies in patients are needed to determine the safety and efficacy of heat therapy in preventing AD.

Heat therapy: possible benefits for cognitive function and the aging brain.

Nonacholic fatty liver disease, or hepatic steatosis, is the most common liver disorder affecting the western world and currently has no pharmacologic cure. Thus, many investigations have focused on alternative strategies to treat or prevent hepatic steatosis. Our laboratory has shown that chronic heat treatment (HT) mitigates glucose intolerance, insulin resistance, and hepatic steatosis in rodent models of obesity. Here, we investigate the direct bioenergetic mechanism(s) surrounding the metabolic effects of HT on hepatic mitochondria. Utilizing mitochondrial proteomics and respiratory function assays, we show that one bout of acute HT (42°C for 20 min) in male C57Bl/6J mice ( = 6/group) triggers a hepatic mitochondrial heat shock response resulting in acute reductions in respiratory capacity, degradation of key mitochondrial enzymes, and induction of mitophagy via mitochondrial ubiquitination. We also show that chronic bouts of HT and recurrent activation of the heat shock response enhances mitochondrial quality and respiratory function via compensatory adaptations in mitochondrial organization, gene expression, and transport even during 4 weeks of high-fat feeding ( = 6/group). Finally, utilizing a liver-specific heat shock protein 72 (HSP72) knockout model, we are the first to show that HSP72, a protein putatively driving the HT metabolic response, does not play a significant role in the hepatic mitochondrial adaptation to acute or chronic HT. However, HSP72 is required for the reductions in blood glucose observed with chronic HT. Our data are the first to suggest that chronic HT (1) improves hepatic mitochondrial respiratory efficiency via mitochondrial remodeling and (2) reduces blood glucose in a hepatic HSP72-dependent manner.

Article	Year
Decoding the Role of Extracellular Vesicles in Liver Diseases. Liver research\| PubMed ID: 29552373	2017
Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis. The Journal of biological chemistry\| PubMed ID: 29666185	2018
Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Li... The American journal of pathology\| PubMed ID: 31026418	2019
Dual Roles of Mammalian Target of Rapamycin in Regulating Liver Injury and Tumorigenesis in Autophagy-Defective Mouse Liver. Hepatology (Baltimore, Md.)\| PubMed ID: 31095752	2019
A review on protein markers of exosome from different bio-resources and the antibodies used for characterization. Journal of histotechnology\| PubMed ID: 31432761	2019
Heat therapy: possible benefits for cognitive function and the aging brain. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 32969779	2020
Heat Treatment Improves Hepatic Mitochondrial Respiratory Efficiency via Mitochondrial Remodeling. Function (Oxford, England)\| PubMed ID: 33629069	2021

Fengyan Deng

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