Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and deadly cancer. Most cases of HCC arise in a cirrhotic/fibrotic liver, indicating that environment may play a paramount role in cancer genesis. Previous studies from our group and others have shown that, in desmoplastic cancers, there is a rich intercellular communication between activated, cancer-associated fibroblasts and cancer cells. Moreover, extracellular vesicles (EVs), or exosomes, have been identified as an important arm of this intercellular communication platform. Finally, these studies have shown that EVs can carry microRNA (miR) species in vivo and deliver them to desmoplastic cancers. The precise role played by activated liver fibroblasts/stellate cells in HCC development is insufficiently known. Based on previous studies, it appears plausible that activated fibroblasts produce signals carried by EVs that promote HCC genesis. In the current study, we first hypothesized and then demonstrated that stellate cell-derived EVs 1) can be loaded with an miR species of choice (miR-335-5p); 2) are taken up by HCC cells in vitro and more importantly in vivo; 3) can supply the miR-335-5p cargo to recipient HCC cells in vitro as well as in vivo; and 4) inhibit HCC cell proliferation and invasion in vitro as well as induce HCC tumor shrinkage in vivo. Finally, we identified messenger RNA targets for miR-335 that are down-regulated after treatment with EV-miR-335-5p. This study informs potential therapeutic strategies in HCC, whereby stellate cell-derived EVs are loaded with therapeutic nucleic acids and delivered in vivo. (Hepatology 2018;67:940-954).

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos).

BACKGROUND AND AIMS: A simple, safe, targeted, and efficient in vivo DNA delivery system is necessary for clinical-grade liver-targeted gene therapy in humans. Intravascular hydrodynamic gene delivery has been investigated in large animal models, but translation to humans has been hampered by its technical challenges, invasiveness, and potential for significant cardiovascular adverse events. We posited that intrabiliary delivery of DNA plasmids via ERCP-guided hydrodynamic injection could overcome these obstacles. METHODS: Twelve pigs (40-50 kg) were divided into 3 groups (4 per group) and survived 21, 30, or 60 days. ERCP was performed by inflating a balloon catheter in the common hepatic duct and creating a closed space between it and the liver parenchyma. Last, a solution composed of plasmid/sleeping beauty (SB) mix was injected under pressure through the catheter into the closed space. Swine were killed at the 3 different time points and liver tissue harvested. Plasmid DNA expression and functional translated protein expression were assessed. RESULTS: ERCP-guided hydrodynamic delivery of naked plasmid DNA facilitated by pCytomegalovirus-Sleep Beauty (pCMV-SB) transposons was technically feasible and devoid of cardiovascular and local adverse events in all 12 pigs. Furthermore, plasmid DNA (both single and combination) was successfully transferred into swine hepatocytes in all 12 pigs. Additionally, stable integration of the DNA constructs in hepatocyte genomic DNA was reliably noted at all 3 time points. In the 4 swine that were kept alive to 60 days, successful genomic integration and subsequent protein expression was observed in the targeted liver tissue. CONCLUSIONS: ERCP-guided hydrodynamic delivery of gene therapy may usher in the next chapter in gene therapy with the potential to impact a variety of single-gene, complex genetic, and epigenetic liver diseases. It also raises the possibility that other nucleic acid therapeutics (microRNA, lncRNA, siRNA, shRNA) could similarly be delivered.

MicroRNAs in the biology and diagnosis of cholangiocarcinoma.

Ever since their discovery, microRNAs (miRNAs) have been the subject of intense investigation of their roles in cells and tissues, both normal and disease state. Although some of the precise mechanisms of biogenesis and actions of miRNAs remain debatable, the fact that miRNAs are dysregulated in diseases such as cancer is undisputed. For many miRNA species, computational databases predict often numerous targets; however, experimental verification in vitro and in vivo is still lacking. For some miRNAs, species-specific targets have been validated; nevertheless, the precise mechanisms those targets act in and whether they are the only truly important ones remain to be discovered. The authors take a closer look at the current status of the role of miRNAs in the diagnosis and biology of cholangiocarcinomas where the perhaps biggest impact in the short term comes from the use of biomarkers in the early diagnosis.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase - Identification of three new potential substrate-interaction sites.

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450. Imidazole is confirmed as a competitive inhibitor with regard to peroxide binding. In contrast, bulky substrates such as anthraquinone dyes are converted at the enzyme surface. In the crystal structure a substrate analog, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), binds to a tyrosine-rich hollow harboring Y25, Y147, and Y337. Spin trapping with a nitric oxide donor uncovers Y229 as an additional tyrosine-based radical center in AauDyPI. Multi-frequency EPR spectroscopy further reveals the presence of at least one intermediate tryptophanyl radical center in activated AauDyPI with W377 as the most likely candidate.

Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated.

Network analysis of a Pkd1-mouse model of autosomal dominant polycystic kidney disease identifies HNF4α as a disease modifier.

Autosomal Dominant Polycystic Kidney Disease (ADPKD; MIM ID's 173900, 601313, 613095) leads to end-stage kidney disease, caused by mutations in PKD1 or PKD2. Inactivation of Pkd1 before or after P13 in mice results in distinct early- or late-onset disease. Using a mouse model of ADPKD carrying floxed Pkd1 alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that Pkd1-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4α as a likely network node. These results are further supported by a meta-analysis of 1,114 published gene expression arrays in Pkd1 wild-type tissues. These analyses also predict that metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that Pkd1 cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating Hnf4α and Pkd1. The Pkd1/Hnf4α double mutants have significantly more cystic kidneys, thus indicating that metabolic pathways could play a role in Pkd1-cystogenesis.

First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer.

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a ß-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits.

Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.

Renal cyst growth is the main determinant for hypertension and concentrating deficit in Pkd1-deficient mice.

We have bred a Pkd1 floxed allele with a nestin-Cre expressing line to generate cystic mice with preserved glomerular filtration rate to address the pathogenesis of complex autosomal dominant polycystic kidney disease (ADPKD) phenotypes. Hypertension affects about 60% of these patients before loss of renal function, leading to significant morbimortality. Cystic mice were hypertensive at 5 and 13 weeks of age, a phenotype not seen in noncystic controls and Pkd1-haploinsufficient animals that do not develop renal cysts. Fractional sodium excretion was reduced in cystic mice at these ages. Angiotensinogen gene expression was higher in cystic than noncystic kidneys at 18 weeks, while ACE and the AT1 receptor were expressed in renal cyst epithelia. Cystic animals displayed increased renal cAMP, cell proliferation, and apoptosis. At 24 weeks, mean arterial pressure and fractional sodium excretion did not significantly differ between the cystic and noncystic groups, whereas cardiac mass increased in cystic mice. Renal concentrating deficit is also an early finding in ADPKD. Maximum urine osmolality and urine nitrite excretion were reduced in 10-13- and 24-week-old cystic mice, deficits not found in haploinsufficient and noncystic controls. A trend of higher plasma vasopressin was observed in cystic mice. Thus, cyst growth most probably plays a central role in early-stage ADPKD-associated hypertension, with activation of the intrarenal renin-angiotensin system as a key mechanism. Cyst expansion is also likely essential for the development of the concentrating deficit in this disease. Our findings are consistent with areas of reduced perfusion in the kidneys of patients with ADPKD.

Polycystin-1 regulates the stability and ubiquitination of transcription factor Jade-1.

Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.

Impaired glomerulogenesis and endothelial cell migration in Pkd1-deficient renal organ cultures.

The PKD1 gene is essential for a number of biological functions, and its loss-of-function causes autosomal dominant polycystic kidney disease (ADPKD). The gene is developmentally regulated and believed to play an essential role in renal development. Previous studies have shown that manipulating murine renal organ cultures with dominant-negative forms of the Pkd1 gene impaired ureteric bud (UB) branching. In the current study, we analyzed different stages of renal development in two distinct mouse models carrying either a null mutation or inactivation of the last two exons of Pkd1. Surprisingly, metanephric explants from Pkd1-deleted kidneys harvested at day E11.5 did not show defects of UB branching and elongation, estimated by cytokeratin staining on fixed tissues or by Hoxb7-GFP time-lapse imaging. However, renal explants from Pkd1-mutants isolated at day E14.5 showed impaired nephrogenesis. Notably, we observed cell migratory defects in the developing endothelial compartment. Previous studies had implicated the Pkd1 gene in controlling cell migration and collagen deposition through PI3 kinases. In line with these studies, our results show that wild-type explants treated with PI3-kinase inhibitors recapitulate the endothelial defects observed in Pkd1 mutants, whereas treatment with VEGF only partially rescued the defects. Our data are consistent with a role for the Pkd1 gene in the endothelium that may be required for proper nephrogenesis.

Radical formation on a conserved tyrosine residue is crucial for DyP activity.

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates. This amino acid is conserved in eucaryotic DyPs but is missing in the structurally related chlorite dismutases (Clds). Dye-decolorizing peroxidases of procaryotic origin equally possess a conserved tyrosine in the same region of the polypeptide albeit not at the homologous position.

Polycystin-1 is required for stereocilia structure but not for mechanotransduction in inner ear hair cells.

The polycystic kidney disease-1 (Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereocilia in vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilli in vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.

Ectopic expression of Cux1 is associated with reduced p27 expression and increased apoptosis during late stage cyst progression upon inactivation of Pkd1 in collecting ducts.

Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid-filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21, and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1(CD) mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1(CD) mouse models of PKD.

Rapamycin ameliorates PKD resulting from conditional inactivation of Pkd1.

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron.

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H(2)O(2) for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 A resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 A resolution by molecular replacement.

Essential role of cleavage of Polycystin-1 at G protein-coupled receptor proteolytic site for kidney tubular structure.

Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis-autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo, we generated by gene targeting a Pkd1 knockin mouse (Pkd1(V/V)) that expresses noncleavable PC1. The Pkd1(V/V) mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo.

Pkd1 haploinsufficiency increases renal damage and induces microcyst formation following ischemia/reperfusion.

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1(+/-) and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploinsufficient mice, a process that apparently depends on a relative deficiency of p21 activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.

Inactivation of Pkd1 in principal cells causes a more severe cystic kidney disease than in intercalated cells.

Renal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1-2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease.