Hepatocyte-targeted siTAZ therapy lowers liver fibrosis in NASH diet-fed chimeric mice with hepatocyte-humanized livers.

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted small interfering RNA (siRNA), GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Following ablation of endogenous hepatocytes, male mice were reconstituted with human hepatocytes from a single donor with the rs738409-C/G PNPLA3 risk variant, resulting in ∼95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient l-amino acid-defined diet for 6 weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. GalNAc-siTAZ lowered human hepatic TAZ and IHH, a TAZ target that promotes NASH fibrosis. Most important, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression versus GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Characterization and Prevention of Hypovitaminosis C in Chimeric Mice with Humanized Livers.

Humanized liver chimeric mice (PXB-mice) are generated by the transplantation of human hepatocytes into mice that have severe combined immunodeficiency and express an albumin-promoted urokinase-type plasminogen activator (cDNA-uPA/SCID) transgene. Human hepatocytes cannot synthesize ascorbic acid (AA; commonly called vitamin C) and humans require supplementation to prevent vitamin C deficiency. PXB-mouse livers contain up to approximately 95% human hepatocytes, which likely affects AA synthesis. To determine whether dietary AA supplementation prevents scurvy-like symptoms and death in PXB-mice, a 12 week study that compared nonsupplemented and supplemented PXB-mice was conducted. Approximately 4 weeks into the study, PXB-mice without dietary supplementation of AA displayed weight loss and clinical signs of hypovitaminosis C, including hunched posture, unkempt appearance, and lameness. Pathologic evaluation of nonsupplemented PXB-mice revealed lesions consistent with hypovitaminosis C. Mean serum AA concentrations in the nonsupplemented PXB-mice were below the limit of quantitation (0.5 µg/mL) and were substantially less than those of controls. AA was also measured in a number of tissues, including adrenal gland, brain, liver, and testis; low AA concentrations were similarly observed in tissues obtained from the nonsupplemented PXB-mice. Collectively, these findings support AA supplementation in PXB-mice to prevent the development of hypovitaminosis C and the potential utility of nonsupplemented PXB-mice as an animal model of scurvy.

Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy.

Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the ß1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research.

The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.

Decreased physical performance is a well-known consequence of rapid ascent to high altitude. Hypoxic pulmonary vasoconstriction (HPV) potentially limits cardiac output and systemic blood flow, thus preventing successful adaptation to rapid ascent. We hypothesized that pharmacological enhancement of the heart rate with theophylline, combined with reversal of HPV via endothelin blockade, could increase exercise performance at high altitude. Female Sprague-Dawley rats were treated with combinations of 1) theophylline, 2) the endothelin receptor antagonists sitaxsentan/ambrisentan, and/or 3) phosphodiesterase-5 inhibitor sildenafil and exposed to either a simulated high altitude (4,267 m) or 12% oxygen. Exercise capacity, peripheral blood flow, hemodynamics, and pulmonary leak were examined. Combination treatment with theophylline and endothelin blockade, but not with the respective single compounds, significantly prolonged run-to-fatigue time under simulated high altitude. No such efficacy was found when theophylline was combined with sildenafil. Neither theophylline nor sitaxsentan or their combination influenced breathing rates and hemoglobin oxygen saturation. Whereas under hypoxia, theophylline significantly increased muscular blood flow, and sitaxsentan increased tissue oxygenation, the combination improved both parameters but in a reduced manner. Under hypoxia, the combination treatment but not the single compounds significantly enhanced pulmonary arterial pressure compared with controls (13.1 ± 6.3 vs. 11.9 ± 5.2 mmHg), whereas mean arterial pressure remained unaffected. Pulmonary wet-to-dry weight ratios were unaffected by combination treatment. We conclude that concomitant dosing with a cardiac stimulant and endothelin antagonist can partially reverse loss of physical performance capacity under hypobaric hypoxia, independent from improving blood oxygen saturation.

Trefoil factor 1 acts to suppress senescence induced by oncogene activation during the cellular transformation process.

Senescence is a cellular stress response characterized by persistent cell growth arrest under various stress conditions, including oncogene activation or tumor suppressor loss, which functions as a critical barrier that must be overcome to allow the progression from a precancerous or preinvasive lesion to a malignant tumor. Trefoil factor 1 (TFF1) is a secreted protein involved in maintaining the gastrointestinal epithelium by serving a tumor-suppressive role; however, TFF1 is overexpressed in several types of cancers. Here we report that TFF1 acts as a promoter of tumorigenesis in the context of prostate and pancreatic cancers by suppressing oncogene-induced senescence (OIS). Expression of TFF1 allows human prostate epithelial cells to escape OIS caused by the activated Ras oncogene or by reduced expression of the tumor suppressor PTEN, in part by the involvement of the EGF receptor-mediated pathway and inhibition of the expression of the cell cycle regulator p21. Without intrinsic promitogenic activity TFF1 may act in both autocrine and paracrine manners to enable cells to undergo the initial transformation and expansion against the restrictive microenvironment during early stage tumorigenesis. Taken together, our findings identify TFF1 as a soluble factor designed to act mainly to antagonize the OIS process to accelerate tumorigenesis.

NDRG4 is required for cell cycle progression and survival in glioblastoma cells.

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133(+) (cancer stem cell (CSC)-enriched) and CD133(-) primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G(1) cell cycle arrest followed by apoptosis. The initial G(1) arrest is associated with a decrease in cyclin D1 expression and an increase in p27(Kip1) expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.

Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.

Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of betaig-h3/TGFBI (transforming growth factor, beta-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the betaig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of betaig-h3 expression dramatically reduced metastasis. Mechanistically, betaig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of the integrin alphavbeta5-Src signaling pathway. Thus, cancers associated with overexpression of betaig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients.

Modeling cancer patient populations in mice: complex genetic and environmental factors.

Genetic differences among individuals contribute to differential susceptibility to cancer and, undoubtedly, to variable efficacy and toxicity of pharmacological-based therapeutics. Many of the specific molecular processes involved in human tumorigenesis have been elucidated and accurately modeled in mice. However, the current models used for drug testing do not accurately predict how new treatments will fare in clinical trials. More sophisticated models that treat cancer as a complex disease present within heterogenous patient populations will provide better predictive power to identify patients that may benefit from specific therapies or that may develop potential drug-induced toxicities.