Bio-Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Inducing immune tolerance with dendritic cell-targeting nanomedicines.

Induced tolerogenic dendritic cells are a powerful immunotherapy for autoimmune disease that have shown promise in laboratory models of disease and early clinical trials. In contrast to conventional immunosuppressive treatments, tolerogenic immunotherapy leverages the cells and function of the immune system to quell the autoreactive lymphocytes responsible for damage and disease. The principle techniques of isolating and reprogramming dendritic cells (DCs), central to this approach, are well characterized. However, the broader application of this technology is limited by its high cost and bespoke nature. Nanomedicine offers an alternative route by performing this reprogramming process in situ. Here, we review the challenges and opportunities in using nanoparticles as a delivery mechanism to target DCs and induce immunomodulation, emphasizing their versatility. We then highlight their potential to solve critical problems in organ transplantation and increasingly prevalent autoimmune disorders such as type 1 diabetes mellitus and multiple sclerosis, where new immunotherapy approaches have begun to show promise.

Epigenetic Compensation Promotes Liver Regeneration.

Two major functions of the epigenome are to regulate gene expression and to suppress transposons. It is unclear how these functions are balanced during physiological challenges requiring tissue regeneration, where exquisite coordination of gene expression is essential. Transcriptomic analysis of seven time points following partial hepatectomy identified the epigenetic regulator UHRF1, which is essential for DNA methylation, as dynamically expressed during liver regeneration in mice. UHRF1 deletion in hepatocytes (Uhrf1HepKO) caused genome-wide DNA hypomethylation but, surprisingly, had no measurable effect on gene or transposon expression or liver homeostasis. Partial hepatectomy of Uhrf1HepKO livers resulted in early and sustained activation of proregenerative genes and enhanced liver regeneration. This was attributed to redistribution of H3K27me3 from promoters to transposons, effectively silencing them and, consequently, alleviating repression of liver regeneration genes, priming them for expression in Uhrf1HepKO livers. Thus, epigenetic compensation safeguards the genome against transposon activation, indirectly affecting gene regulation.

Regulation of fatty acid trafficking in liver by thioesterase superfamily member 1.

Thioesterase superfamily member 1 (Them1) is an acyl-CoA thioesterase that is highly expressed in brown adipose tissue, where it functions to suppress energy expenditure. Lower Them1 expression levels in the liver are upregulated in response to high-fat feeding. Them1-/- mice are resistant to diet-induced obesity, hepatic steatosis, and glucose intolerance, but the contribution of Them1 in liver is unclear. To examine its liver-specific functions, we created conditional transgenic mice, which, when bred to Them1-/- mice and activated, expressed Them1 exclusively in the liver. Mice with liver-specific Them1 expression exhibited no changes in energy expenditure. Rates of fatty acid oxidation were increased, whereas hepatic VLDL triglyceride secretion rates were decreased by hepatic Them1 expression. When fed a high-fat diet, Them1 expression in liver promoted excess steatosis in the setting of reduced rates of fatty acid oxidation and preserved glycerolipid synthesis. Liver-specific Them1 expression did not influence glucose tolerance or insulin sensitivity, but did promote hepatic gluconeogenesis in high-fat-fed animals. This was attributable to the generation of excess fatty acids, which activated PPARα and promoted expression of gluconeogenic genes. These findings reveal a regulatory role for Them1 in hepatocellular fatty acid trafficking.

Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development.

Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC.

Mice with hepatocyte-specific deficiency of type 3 deiodinase have intact liver regeneration and accelerated recovery from nonthyroidal illness after toxin-induced hepatonecrosis.

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.

UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

Fibrinogen like protein 1(Fgl1) is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.