Nikita Deo

BACKGROUND: High levels of Lp(a) (lipoprotein(a)) are associated with multiple forms of cardiovascular disease. Lp(a) consists of an apoB ₁₀₀ -containing particle attached to the plasminogen homologue apo(a). The pathways for Lp(a) clearance are not well understood. We previously discovered that the plasminogen receptor PlgRKT (plasminogen receptor with a C-terminal lysine) promoted Lp(a) uptake in liver cells. Here, we aimed to further define the role of PlgRKT and to investigate the role of 2 other plasminogen receptors, annexin A2 and S100A10 (S100 calcium-binding protein A10) in the endocytosis of Lp(a). METHODS: Human hepatocellular carcinoma (HepG2) cells and haploid human fibroblast-like (HAP1) cells were used for overexpression and knockout of plasminogen receptors. The uptake of Lp(a), LDL (low-density lipoprotein), apo(a), and endocytic cargos was visualized and quantified by confocal microscopy and Western blotting. RESULTS: The uptake of both Lp(a) and apo(a), but not LDL, was significantly increased in HepG2 and HAP1 cells overexpressing PlgRKT, annexin A2, or S100A10. Conversely, Lp(a) and apo(a), but not LDL, uptake was significantly reduced in HAP1 cells in which PlgRKT and S100A10 were knocked out. Surface binding studies in HepG2 cells showed that overexpression of PlgRKT, but not annexin A2 or S100A10, increased Lp(a) and apo(a) plasma membrane binding. Annexin A2 and S100A10, on the other hand, appeared to regulate macropinocytosis with both proteins significantly increasing the uptake of the macropinocytosis marker dextran when overexpressed in HepG2 and HAP1 cells and knockout of S100A10 significantly reducing dextran uptake. Bringing these observations together, we tested the effect of a PI3K (phosphoinositide-3-kinase) inhibitor, known to inhibit macropinocytosis, on Lp(a) uptake. Results showed a concentration-dependent reduction confirming that Lp(a) uptake was indeed mediated by macropinocytosis. CONCLUSIONS: These findings uncover a novel pathway for Lp(a) endocytosis involving multiple plasminogen receptors that enhance surface binding and stimulate macropinocytosis of Lp(a). Although the findings were produced in cell culture models that have limitations, they could have clinical relevance since drugs that inhibit macropinocytosis are in clinical use, that is, the PI3K inhibitors for cancer therapy and some antidepressant compounds.

Serotonin is a neurotransmitter and a hormone that is typically associated with regulating our mood. However, the serotonin transporter and receptors are expressed throughout the body, highlighting the much broader, systemic role of serotonin in regulating human physiology. A substantial body of data strongly implicates serotonin as a fundamental regulator of endocytosis and endocytic sorting. Serotonin has the potential to enhance endocytosis through three distinct mechanisms – serotonin signalling, serotonylation and insertion into the plasma membrane – although the interplay and relationship between these mechanisms has not yet been explored. Endocytosis is central to the cellular response to the extracellular environment, controlling receptor distribution on the plasma membrane to modulate signalling, neurotransmitter release and uptake, circulating protein and lipid cargo uptake, and amino acid internalisation for cell proliferation. Uncovering the range of cellular and physiological circumstances in which serotonin regulates endocytosis is of great interest for our understanding of how serotonin regulates mood, and also the fundamental understanding of endocytosis and its regulation throughout the body.This article has an associated Future Leader to Watch interview with the first author of the paper.

We recently found that plasminogen receptors regulate the plasma membrane binding and uptake of Lp(a) via macropinocytosis. In this study, we sought to further define lipoprotein(a) [Lp(a)] macropinocytosis, discovering an unexpected role for antidepressants and serotonin in the regulation of this process. We found that the tricyclic antidepressant imipramine enhanced Lp(a) uptake, in contradiction of its published role as a macropinocytosis inhibitor. Extending these experiments to the commonly used serotonin uptake inhibitors (SSRIs) citalopram and sertraline, we found that citalopram stimulated, while sertraline inhibited, Lp(a) uptake. Imipramine and citalopram enhanced cell surface binding of Lp(a) rather than upregulating macropinocytosis. Consistent with imipramine and citalopram boosting extracellular serotonin levels, serotonin itself also enhanced Lp(a) surface binding and uptake. Imipramine and serotonin increased expression of the plasminogen receptor with a C-terminal lysine (PlgRKT), a receptor known to enhance cell surface binding of Lp(a), likely accounting for their effects on Lp(a) uptake. Finally, imipramine and citalopram increased Lp(a) delivery into Rab11 recycling endosomes, but not degradative pathways in the cell. These findings indicate citalopram and imipramine may have utility as a potential Lp(a)-lowering therapeutic in people suffering from depression who often have elevated Lp(a) levels and an increased risk of cardiovascular disease.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Tibial pseudarthrosis often features deficient bone formation, excessive bone resorption, and extensive pathological fibrosis, particularly in individuals with Neurofibromatosis type I (NF1). It was hypothesized that overactive NF1-Ras-JNK signalling may underlie the pathological fibrosis, and that this could be treated via a JNK antagonist. CC-930, a small molecule JNK inhibitor, was trialed in closed fractures in wild type mice CC-930 (25 mg/kg/twice daily) was dosed throughout fracture healing (D2-21) and during the latter stages of repair (D11-21). All fractures healed by D21, regardless of treatment, with some of the CC-930 (D11-21) treatment group showing early bridging. CC-930 (D11-21) was tested in an Nf1-null fracture model where Nf1 was inactivated by Ad-Cre virus injection in Nf1 flox/flox mice; these mice also possessed a Cre-responsive tdTomato transgene. CC-930 resulted in a significant decrease in non-unions (93% vehicle vs. 64% CC-930, p < 0.01). Local treatment with the bone anabolic rhBMP-2 (10 mg) increased union and callus bone volume, but also increased the fibrotic tissue at the fracture site. Fractures treated with a combination of rhBMP-2 (10 mg) and CC-930 were all partially or fully bridged by D21 (p < 0.01 vs. vehicle) and there was a significant decrease in fibrosis vs. rhBMP-2 alone (p < 0.01). In untreated Nf1null fractures, the tdTomato transgene was expressed in fibrous tissue at the fracture site, but not in the newly forming bone. These data suggest that JNK inhibition may be an effective therapeutic approach for reducing pathological fibrosis in NF1 tibial pseudarthrosis, however other adjunctive strategies may be required to augment bone formation.

Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un‐treated control fractures (7% union). In contrast, rhBMP‐2 and rhBMP‐2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP‐2 led to a 12‐fold increase in callus bone volume and this was further increased in the rhBMP‐2/ZA group. Mechanical testing of the healed rhBMP‐2 and rhBMP‐2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un‐fractured contralateral leg. Co‐treatment with rhBMP‐2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti‐resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:930–936, 2018.

As-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy. Methods: In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1−/− calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 μg), or a combination of rhBMP-2 and PD0325901. Results: Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced. Conclusions: The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis. Clinical Relevance: This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis....