Citations

Vascular endothelial growth factor receptor‐2 (VEGFR2) plays a key role in vasculogenesis and pathophysiological angiogenesis. Cell surface levels of VEGFR2 is regulated by endocytosis and secretory transport of newly synthesized receptors to the plasma membrane. The mechanism of VEGFR2 sorting and trafficking along the secretory pathway is not well defined. In the present study, using human endothelial cells we demonstrate that sorting and trafficking of VEGFR2 from the Golgi is regulated by syntaxin 6, a t‐SNARE involved in intra‐Golgi and post‐Golgi membrane fusion events. Loss of syntaxin 6 function results in altered sorting of VEGFR2 from the Golgi resulting in receptor down regulation. Our data support a model in which syntaxin 6 regulates delivery of VEGFR2 from the Golgi apparatus to the cell surface thereby regulating VEGF‐A‐induced endothelial cell proliferation, migration, and morphogenesis. Grant Funding Source NIH

AbstractIgA nephropathy (IgAN), an immune complex‐mediated process and the most common primary glomerulonephritis, can progress to end‐stage renal disease in up to 40% of patients. Accordingly, a therapeutic strategy targeting a specific molecular pathway is urgently warranted. Aided by structure characterisation and target identification, we predicted that a novel ring‐fused 6‐(2,4‐difluorophenyl)‐3‐(3‐(trifluoromethyl)phenyl)‐2H‐benzo[e][1,3]oxazine‐2,4(3H)‐dione (LCC18) targets the NLRP3 inflammasome, which participates in IgAN pathogenesis. We further developed biomarkers for the disease. We used two complementary IgAN models in C57BL/6 mice, involving TEPC‐15 hybridoma‐derived IgA, and in gddY mice. Moreover, we created specific cell models to validate therapeutic effects of LCC18 on IgAN and to explain its underlying mechanisms. IgAN mice benefited significantly from treatment with LCC18, showing dramatically improved renal function, including greatly reduced proteinuria and renal pathology. Mechanistic studies showed that the mode of action specifically involved: (1) blocking of the MAPKs/COX‐2 axis‐mediated priming of the NLRP3 inflammasome; (2) inhibition of ASC oligomerisation and NLRP3 inflammasome assembly by inhibiting NLRP3 binding to PKR, NEK7 and ASC; and (3) activation of autophagy. LCC18 exerts therapeutic effects on murine IgAN by differentially regulating NLRP3 inflammasome activation and autophagy induction, suggesting this new compound as a promising drug candidate to treat IgAN. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Sprouting angiogenesis is a highly coordinately process controlled by vascular endothelial growth factor receptor (VEGFR)‐Notch signaling. Here we investigated whether Tripartite motif‐containing 28 (TRIM28), which is an epigenetic modifier implicated in gene transcription and cell differentiation, is essential to mediate sprouting angiogenesis. We observed that knockdown of TRIM28 ortholog in zebrafish resulted in developmental vascular defect with disorganized and reduced vasculatures. Consistently, TRIM28 knockdown inhibited angiogenic sprouting of cultured endothelial cells (ECs), which exhibited increased mRNA levels of VEGFR1, Delta‐like (DLL) 3, and Notch2 but reduced levels of VEGFR2, DLL1, DLL4, Notch1, Notch3, and Notch4.The regulative effects of TRIM28 on these angiogenic factors were partially mediated by hypoxia‐inducible factor 1 α (HIF‐1α) and recombination signal‐binding protein for immunoglobulin kappa J region (RBPJκ). In vitro DNA‐binding assay showed that TRIM28 knockdown increased the association of RBPJκ with DNA sequences containing HIF‐1α‐binding sites. Moreover, the phosphorylation of TRIM28 was controlled by VEGF and Notch1 through a mechanism involving RBPJκ‐dual‐specificity phosphatase (DUSP)‐p38 MAPK, indicating a negative feedback mechanism. These findings established TRIM28 as a crucial regulator of VEGFR‐Notch signaling circuit through HIF‐1α and RBPJκ in EC sprouting angiogenesis.

Significance Statement The arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis, but several studies have shown that the primary patency rate at 1 year is approximately 60%. Percutaneous transluminal angioplasty (PTA) is the first line of treatment for AVF stenosis. However, the rate of restenosis after angioplasty is high and the primary patency rate after PTA treatment in AVFs at 6, 12, and 24 months is 61%, 42%, and 35%, respectively. Currently, no durable therapies prolong patency after angioplasty. This paper describes the use of adipose-derived mesenchymal stem cells to reduce stenosis after PTA treatment in a murine AVF model and provides rationale for potential clinical use. Background Percutaneous transluminal angioplasty (PTA) is the first line of treatment for stenosis in the arteriovenous fistula (AVF) created to provide access for hemodialysis, but resenosis still occurs. Transplants of adipose-derived mesenchymal stem cells (AMSCs) labeled with green fluorescent protein (GFP) to the adventitia could reduce pro-inflammatory gene expression, possibly restoring patency in a murine model of PTA for venous stenosis. Methods Partial nephrectomy of male C57BL/6J mice induced CKD. Placement of the AVF was 28 days later and, 14 days after that, PTA of the stenotic outflow vein was performed with delivery of either vehicle control or AMSCs (5×10 5 ) to the adventitia of the vein. Mice were euthanized 3 days later and gene expression for interleukin-1 beta (IL-1 β ) and tumor necrosis factor-alpha TNF- α ) analyzed, and histopathologic analysis performed on day 14 and 28. GFP (+) AMSCs were tracked after transplantation for up to 28 days and Doppler ultrasound performed weekly after AVF creation. Results Gene and protein expression of IL-1 β and TNF- α, fibrosis, proliferation, apoptosis and smooth muscle actin decreased, and the proportions of macrophage types (M2/M1) shifted in a manner consistent with less inflammation in AMSC-transplanted vessels compared to controls. After PTA, AMSC-treated vessels had significantly higher wall shear stress, average peak, and mean velocity, with increased lumen vessel area and decreased neointima/media area ratio compared to the control group. At 28 days after delivery, GFP (+) AMSC were present in the adventitia of the outflow vein. Conclusions AMSC-treated vessels had improved vascular remodeling with decreased proinflammatory gene expression, inflammation, and fibrotic staining compared to untreated vessels.

Despite recent advancements, effective treatment for pancreatic ductal adenocarcinoma (PDAC) has remained elusive. The overall survival rate in PDAC patients has been dismally low due to resistance to standard therapies. In fact, the failure of monotherapies to provide long-term survival benefits in patients led to ascension of several combination therapies for PDAC treatment. However, these combination therapies provided modest survival improvements while increasing treatment-related adverse side effects. Hence, recent developments in drug delivery methods hold the potential for enhancing therapeutic benefits by offering cocktail drug loading and minimizing chemotherapy-associated side effects. Nanoformulations-aided deliveries of anticancer agents have been a success in recent years. Yet, improving the tumor-targeted delivery of drugs to PDAC remains a major hurdle. In the present paper, we developed several new tumor-targeted dual intervention-oriented drug-encapsulated (DIODE) liposomes. We successfully formulated liposomes loaded with gemcitabine (G), paclitaxel (P), erlotinib (E), XL-184 (c-Met inhibitor, X), and their combinations (GP, GE, and GX) and evaluated their in vitro and in vivo efficacies. Our novel DIODE liposomal formulations improved median survival in comparison with gemcitabine-loaded liposomes or vehicle. Our findings are suggestive of the importance of the targeted delivery for combination therapies in improving pancreatic cancer treatment.

Abstract Like VEGF, morphine stimulates MAPK/ERK and Akt, leading to the promotion of angiogenesis via NO dependent signaling (Cancer Res62: 4491, 2002). Morphine acts via pertussis toxin (PT)-dependent G-protein coupled receptors (GPCRS), while VEGF acts via receptor tyrosine kinases (RTKs). We showed that PT-dependent GPCRs transactivate VEGF receptor-2/Flk1 via small GTPase RhoA (JBC277: 4679, 2002; JBC278:20738, 2003). Therefore, we hypothesized that morphine via the mu opioid receptor (MOR) transactivates Flk1 and promotes a pro-angiogenic microenvironment. Morphine-induced proliferation of human umbilical vein endothelial cells (HUVEC) was completely abrogated by Y-27632 (100 μM), a highly selective and potent inhibitor of Rho-associated protein kinases, suggesting the activation of Rho signaling by morphine. Addition of 1 μM morphine potentiated VEGF-induced (10 ng/ml) proliferation of HUVEC by 25%. We observed a 30% increase in intracellular calcium release after VEGF stimulation of HUVEC pre-incubated with morphine as compared to HUVEC pre-incubated with PBS, detected by a change in the fluorescence ratio of the Fura-2 AM dye. These findings show that morphine, via MOR and Rho signaling, transactivates Flk1 leading to the stimulation of calcium signaling and endothelial cell proliferation. To functionally corroborate our hypothesis, we used MOR knockout (MOR-KO) mice and injected them with MOR-replete T241 fibrosarcoma cells. T241 fibrosarcoma tumor growth in vivo showed appearance of palpable and measurable tumors 2 days earlier in wild type (wt) as compared to MOR-KO mice. Tumor growth and angiogenesis were decreased by 20–35% in MOR-KO mice as compared to wt littermates during 3 weeks of tumor growth. None of the MOR-KO showed signs of lung metastasis versus 40% wt mice with metastasis. Morphine (1.42 for the first 2 wks and 2.14 mg/Kg/day later, respectively) stimulated 20–35% tumor growth in wt, but not in MOR-KO mice. Western immunoblotting showed a 10-fold increase in the expression of phospho-Flk1 in morphine treated wt tumors as compared to PBS-treated wt mice. Morphine did not stimulate phospho-Flk1 expression in MOR-KO mice. Western analysis of immunoprecipitates obtained with α-MOR antibody showed the expression of Flk1 and phospho-Flk1 in wt, but were not expressed in MOR-KO tumors. Thus, MOR stimulates the transactivation of Flk1 in wt mice but not in MOR-KO. These in vitro and in vivo data using MOR-KO mice and the MOR agonist, morphine, show that MOR stimulates endothelial proliferation, angiogenesis and promotes tumor growth and metastasis directly as well as by transactivating Flk1 phosphorylation. We speculate that MOR is a critical component of the ‘angiogenic switch’, which regulates the pro-angiogenic and growth promoting tumor microenvironment. Thus, MOR provides a novel target for developing anti-angiogenic therapies.

Abstract Background: B-Chronic Lymphocytic Leukemia (CLL) is predominantly characterized as a clonal B-cell disorder where a minor fraction of CLL B cells proliferate, but the majority of these cells are non-cycling. Importantly, CLL B cells also exhibit apoptosis resistance that results from a complex set of redundant mechanisms. This latter feature presents a significant problem in relation to disease progression and developing effective therapies in CLL. Previously, we and others have reported that leukemic CLL cells spontaneously secrete pro-angiogenic vascular endothelial growth factor (VEGF) that can significantly enhance CLL leukemic B cell apoptosis resistance via unknown mechanisms. In addition, the molecular mechanism of the constitutive upregulation of VEGF in CLL B cells is unknown. Methods and Results: Here, we report that CLL B cells express a high level of HIF-1α protein under normoxic conditions as compared to that of normal B cells in Western blot analysis using a specific antibody. Accumulation of HIF-1α in CLL B cells resulted in upregulation of its downstream target VEGF. To comprehend this abnormal overexpression of HIF-1α, we have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation by Western blot analysis and found it to be either absent or notably low level in CLL B cells as compared to that of normal B cells. Furthermore, we have shown by in vitro reporter gene assays that miR-92a, a microRNA known to be overexpressed in CLL B cells, can target the VHL 3′-untranslated region (UTR) and is likely a reason for the depressed levels of pVHL in CLL B cells. To validate our hypothesis, we transiently transfected the human embryonic kidney cell line (293T) with miR-92a and observed a dose-dependent repression of pVHL level. Post-transcriptional regulation of the VHL gene was further confirmed when we observed a subtle but definite increase of the pVHL levels monitored by immunoblot following nucleofection of the antisense oligo targeted to miR-92a into primary CLL B cells. To examine whether HIF-1α is functionally active in CLL B cells, we performed co-immunoprecipitation experiments and found that HIF-1α forms an active complex by physical association with the transcriptional co-activator p300 and phospho-STAT3 in CLL B cells. The latter molecule is known to be constitutively expressed in CLL B cells but its function has been unknown. Subsequently, we have found via chromatin immunoprecipitation analyses in CLL B cells that HIF-1α and STAT3 are bound to the VEGF promoter and also recruit RNA polymerase II, further substantiating that this complex likely functions in the activation of VEGF transcription. Summary: Overall, our data show that CLL B cells express constitutively elevated HIF-1α levels in normoxia due, at least in part, to miR-92a regulated diminished pVHL expression. This is a unique previously undescribed mechanism for enhanced HIF-1α levels in human malignancy. We believe that Increased VEGF expression is associated with the combined effects of HIF-1α, p300 and constitutively active STAT3 as a functional complex and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. These findings now provide additional strategy points for interrupting the VEGF autocrine pathway in CLL B cells.