Citations

Purpose: The majority of patients with end stage renal disease who require hemodialysis use a arteriovenous fistula (AVF) for their vascular access. However, only 40% of AVFs are clinically usable after 1 year. Arteriovenous fistulas fail because of venous neointimal hyperplasia (VNH) that develops in the proximal outflow vein. The immediate early gene, IEX-1 (IER-3), is increased in response to shear stress and hypoxia and both factors contribute to VNH. IEX-1 expression was increased in stenotic venous stenoses removed from patients with hemodialysis vascular access compared to controls. We hypothesized that reducing the expression of IEX-1 using a knockout mouse model with chronic kidney disease (CKD) and AVFs would have decreased VNH compared to wild type (WT) mice. Methods: CKD was induced by performing a nephrectomy. Four weeks later, an AVF was placed to connect the carotid artery to ipsilateral jugular vein. The mice were sacrificed 3 and 28 days after fistula creation and outflow veins were harvested for gene expression and histomorphometric analyses. Results: By day 3, the average gene expression of vascular endothelial growth factor-A (VEGF-A) was significantly lower in the IEX-1 KO vs. WT mice (average reduction: 40%, P<0.05). At day 28, the average lumen vessel area was increased significantly (average increase: 133%, P<0.05, IEX-1 KO vs. WT, respectively) while the average area of the neointima was significantly decreased (average reduction: 20%, IEX-1 KO vs. WT, respectively). Accompanied with this, there was a significant decrease in α-SMA staining and Ki-67 proliferation while the average TUNEL staining was significantly increased (all P<0.05, IEX-1 KO vs. WT). Finally, there was a significant decrease in the average staining intensity for VEGF-A and matrix metalloproteinase-9 (MMP-9) in the KO vs. WT mice (both, P<0.05). Conclusion: A reduction in IEX-1 gene expression results in positive vascular remodeling accompanied with a reduction in proliferation and α-SMA density mediated through a VEGF-A/MMP-9 pathway. Taken collectively, these findings indicate that reducing IEX-1 expression is protective for VNH formation and provide a rationale for use of anti-IEX-1 therapies aimed at prolonging AVF function.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance cloud the outcomes. Hence, there is an urgent need to explore new therapeutic targets and formulate innovative strategies for the more effective treatment of PDAC.Methods: We transduced pancreatic cancer cells with a protein kinase library, and transfected cells were selected by puromycin treatment, followed by continuous exposure to vehicle control or gemcitabine/paclitaxel chemotherapy for 21 days. Next-generation sequencing as well as MAGeCK-VISPR analysis were performed to the identify candidate genes. Finally, we used the CRISPR-Cas9 to knockout the target gene, confirming that it is a viable target for overcoming chemoresistance in vivo and in vitro.Results: We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, cyclin-dependent kinase 7 (CDK7) ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction. Furthermore, THZ1 synergized with gemcitabine and paclitaxel in vitro and in vivo, resulting in enhanced antitumor effects.Conclusions: Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and provide new strategies for overcoming chemoresistance in pancreatic cancer.

Abstract BackgroundEfficacy of targeted drug delivery using nanoparticles relies on several factors including the uptake mechanisms such as phagocytosis, macropinocytosis, micropinocytosis and receptor mediated endocytosis. These mechanisms have been studied with respect to the alteration in signaling mechanisms, cellular morphology, and linear nanomechanical properties (NMPs). Commonly employed classical contact mechanics models to address cellular NMPs fail to address mesh like structure consisting of bilayer lipids and proteins of cell membrane. To overcome this technical challenge, we employed poroelastic model which accounts for the biphasic nature of cells including their porous behavior exhibiting both solid like (fluid storage) and liquid like (fluid dissipate) behavior ResultsIn this study, we employed atomic force microscopy to monitor the influence of surface engineering of gold nanoparticles (GNPs) to the alteration of nonlinear NMPs such as drained Poisson’s ratio, effective shear stress, diffusion constant and pore dimensions of cell membranes during their uptake. Herein, we used pancreatic cancer (PDAC) cell lines including Panc1, AsPC-1 and endothelial cell HUVECs to understand the receptor-dependent and -independent endocytosis of two different GNPs derived using plectin-1 targeting peptide (PTP-GNP) and corresponding scrambled peptide (sPEP-GNP). Compared to untreated cells, in case of receptor dependent endocytosis of PTP-GNPs diffusion coefficient altered ~1264-fold and ~1530-fold and pore size altered ~320-fold and ~260-fold in Panc1 and AsPC-1 cells respectively. Whereas for receptor independent mechanisms, we observed modest alteration in diffusion coefficient and pore size, in these cells compared to untreated cells. Effective shear stress corresponding to 7.38±0.15 kPa and 20.49±0.39 kPa in PTP-GNP treatment in Panc1 and AsPC-1, respectively was significantly more than that for sPEP-GNP. These results demonstrate that with temporal recruitment of plectin-1 during receptor mediated endocytosis affects the poroelastic attributes of the membrane. ConclusionThis study confirms that nonlinear NMPs of cell membrane are directly associated with the uptake mechanism of nanoparticles and can provide promising insights of the nature of endocytosis mechanism involved for organ specific drug delivery using nanoparticles. Hence, nanomechanical analysis of cell membrane es using this noninvasive, label-free and live-cell analytical tool can therefore be instrumental to evaluate therapeutic benefit of nanoformulations.

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status.

Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein–coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine- N -oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.

The evidence that regular physical exercise reduces the risk of developing cancer is well described. However, the interaction between physical exercise and cancer is not fully clarified yet. Several myokines released by skeletal muscle appear to have a direct anti-tumour function. There are few data on myokine secretion after exercise in patients with advanced tumours. Pancreatic cancer (PC) is a very aggressive and usually fatal cancer. To investigate the effects of exercise in PC, the blood of advanced-stage PC patients was analysed after 12 weeks of resistance training using whole-body electromyostimulation. After the 12-week training period, the patient serum inhibited the proliferation and the motility of PC cells and enhanced PC cell apoptosis. The impact of exercise training was also investigated in an exercise-mimicking in vitro model using electric pulse stimulation of human myotubes and revealed similar anti-tumour effects on PC cells, clearly indicating direct cancer-protective properties of activated skeletal muscle. Protein and gene expression analyses in plasma from exercise-trained patients and in myotube cultures after in vitro exercise showed that interleukin 10 (IL10), C-X-C motif ligand 1 (CXCL1) and C-C motif chemokine ligand 4 (CCL4) are myokines released from activated skeletal muscle. In accordance with the effects of serum from exercise-trained patients, the supplementation with recombinant IL10, CXCL1 and CCL4 impaired growth and migration of PC cells. Treatment of PC cells with these myokines upregulated caspase 3/7 expression and the cleavage of poly(ADP-ribose) polymerase, leading to enhanced PC cell death. The identification of myokines with anti-tumour properties in advanced-stage PC patients after exercise opens a new perspective in supportive therapy with sports and exercise for cancer patients.

Abstract In the present study, acute onset of severe lupus nephritis was successfully treated in mice using a new, benzamide‐linked, small molecule that targets immune modulation and the NLRP3 inflammasome. Specifically, 6‐(2,4‐difluorophenyl)‐3‐(3‐(trifluoromethyl)phenyl)‐2H‐benzo[e][1,3]oxazine‐2,4(3H)‐dione (Cf‐02) (a) reduced serum levels of IgG anti‐dsDNA, IL‐1β, IL‐6, and TNF‐α, (b) inhibited activation of dendritic cells and differentially regulated T cell functions, and (c) suppressed the NF‐κB/NLRP3 inflammasome axis, targeting priming and activating signals of the inflammasome. Moreover, treatment with Cf‐02 significantly inhibited secretion of IL‐1β in lipopolysaccharide‐stimulated macrophages, but this effect was abolished by autophagy induction. These results recommend Cf‐02 as a promising drug candidate for the serious renal conditions associated with systemic lupus erythematosus. Future investigations should examine whether Cf‐02 may also be therapeutic in other types of chronic kidney disease involving NLRP3 inflammasome‐driven signaling.

Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.

Vascular permeability factor/vascular endothelial growth factor A (VEGF) is a central regulator of angiogenesis and potently promotes vascular permeability. VEGF plays a key role in the pathologies of heart disease, stroke, and cancer. Therefore, understanding the molecular regulation of VEGF signaling is an important pursuit. Rho GTPase proteins play various roles in vasculogenesis and angiogenesis. While the functions of RhoA and RhoB in these processes have been well defined, little is known about the role of RhoC in VEGF-mediated signaling in endothelial cells and vascular development. Here, we describe how RhoC modulates VEGF signaling to regulate endothelial cell proliferation, migration and permeability. We found VEGF stimulation activates RhoC in human umbilical vein endothelial cells (HUVECs), which was completely blocked after VEGF receptor 2 (VEGFR-2) knockdown indicating that VEGF activates RhoC through VEGFR-2 signaling. Interestingly, RhoC knockdown delayed the degradation of VEGFR-2 compared to control siRNA treated HUVECs, thus implicating RhoC in VEGFR-2 trafficking. In light of our results suggesting VEGF activates RhoC through VEGFR-2, we sought to determine whether RhoC regulates vascular permeability through the VEGFR-2/phospholipase Cγ (PLCγ) /Ca 2+ /eNOS cascade. We found RhoC knockdown in VEGF-stimulated HUVECs significantly increased PLC-γ1 phosphorylation at tyrosine 783, promoted basal and VEGF-stimulated eNOS phophorylation at serine 1177, and increased calcium flux compared with control siRNA transfected HUVECs. Taken together, our findings suggest RhoC negatively regulates VEGF-induced vascular permeability. We confirmed this finding through a VEGF-inducible zebrafish model of vascular permeability by observing significantly greater vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Furthermore, we showed that RhoC promotes endothelial cell proliferation and negatively regulates endothelial cell migration. Our data suggests a scenario in which RhoC promotes proliferation by upregulating -catenin in a Wnt signaling-independent manner, which in turn, promotes Cyclin D1 expression and subsequently drives cell cycle progression.

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-2 to control vasculogenesis and angiogenesis. Dysregulation of these physiological processes contributes to the pathologies of heart disease, stroke, and cancer. Protein kinase D (PKD) plays a crucial role in the regulation of angiogenesis by modulating endothelial cell proliferation and migration. In human umbilical vein endothelial cells (HUVEC) and human blood outgrowth endothelial cells (BOEC), knockdown of PKD-1 or PKD-2 downregulates VEGFR-2 and significantly inhibits VEGF-induced endothelial cell proliferation and migration. We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, activating enhancer binding protein 2 (AP2) binding sites exist within the VEGFR-2 promoter. Thus, we hypothesized PKD may downregulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. Indeed, AP2β binds the VEGFR-2 promoter upon PKD knockdown in HUVEC as evident by chromatin immunoprecipitation assay. Luciferase reporter assays using serial deletions of AP2β binding sites within the VEGFR-2 promoter revealed transcriptional activity negatively correlated with the number of AP2β binding sites, thus confirming negative regulation of VEGFR-2 transcription by AP2β. Next, using siRNA, we demonstrated that upregulation of AP2β decreased VEGFR-2 expression and loss of AP2β enhanced VEGFR-2 expression. In vivo studies confirmed this finding as we observed increased VEGFR-2 immunostaining in the dorsal horn of the spinal cord of embryonic day 13 AP2β knockout mice. We hypothesize that PKD directly regulates AP2β function by serine phosphorylation and ongoing studies are being conducted to determine phosphorylation sites in AP2β directly regulated by PKD. Taken together, we demonstrate AP2β negatively regulates VEGFR-2 transcription and VEGFR-2 is a major downstream target of PKD. Our findings describing how PKD regulates angiogenesis may contribute to the development of therapies to improve the clinical outcome of patients afflicted by heart disease, stroke, and cancer.