Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signalling molecules bind to NRPs through a carboxy (C)-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumour-homing CendR peptides penetrate through tumour tissue and have shown utility in enhancing drug delivery into tumours. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signalling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway.
There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.
Fibrosis is a deleterious consequence of chronic inflammation in a number of human pathologies ultimately leading to organ dysfunction and failure. Two growth factors that are important in blood vessel physiology and tissue fibrosis, platelet-derived growth factor (PDGF)-B and transforming growth factor (TGF)-?1, were investigated. Adenoviral vectors were used to induce transient overexpression of these growth factors in mouse skin. Changes in tissue structure and protein and mRNA expressions were investigated. Both PDGF-B and TGF-?1 could initiate but neither could sustain angiogenesis. Instead, vascular regression was observed. Overexpression of both TGF-?1 and PDGF-B led to a marked macrophage influx and an expansion of the connective tissue cell population. Over time, this effect was sustained in mice treated with TGF-?1, whereas it was partially reversible in mice treated with PDGF-B. On the basis of structure and expression of phenotypical markers, the emerging connective tissue cell population may originate from microvascular pericytes. TGF-?1 induced expansion of connective tissue cells with a myofibroblast phenotype, whereas PDGF-B induced a fibroblast phenotype negative for ?-smooth muscle actin. TGF-?1 and PDGF-B overexpressions mediated distinct effects on mRNA transcript levels of fibrillar procollagens, their modifying enzymes, small leucin-rich repeat proteoglycans, and matricellular proteins affecting both the composition and the quantity of the extracellular matrix. This study offers new insight into the effects of PDGF-B and TGF-?1 on the vasculature and connective tissue in vivo.
The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 (m1J) mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 (m1J) mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 (m1J) mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.
Microvascular pericytes are of key importance in neoformation of blood vessels, in stabilization of newly formed vessels as well as maintenance of angiostasis in resting tissues. Furthermore, pericytes are capable of differentiating into pro-fibrotic collagen type I producing fibroblasts. The present study investigates the effects of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on pericyte proliferation, cell viability, migration and differentiation. The results show that HDAC inhibition through exposure of pericytes to VPA in vitro causes the inhibition of pericyte proliferation and migration with no effect on cell viability. Pericyte exposure to the potent HDAC inhibitor Trichostatin A caused similar effects on pericyte proliferation, migration and cell viability. HDAC inhibition also inhibited pericyte differentiation into collagen type I producing fibroblasts. Given the importance of pericytes in blood vessel biology a qPCR array focusing on the expression of mRNAs coding for proteins that regulate angiogenesis was performed. The results showed that HDAC inhibition promoted transcription of genes involved in vessel stabilization/maturation in human microvascular pericytes. The present in vitro study demonstrates that VPA influences several aspects of microvascular pericyte biology and suggests an alternative mechanism by which HDAC inhibition affects blood vessels. The results raise the possibility that HDAC inhibition inhibits angiogenesis partly through promoting a pericyte phenotype associated with stabilization/maturation of blood vessels.
How activation of a specific growth factor receptor selectively results in either cell proliferation or cytoskeletal reorganization is of central importance to the field of pathophysiology. In this study, we report on a novel mechanism that explains how this process is accomplished. Our current investigation demonstrates that soluble platelet derived growth factor- (PDGF)-BB activates a cohort of PDGF-beta receptors primarily confined to the lipid raft component of the cell membrane, specifically caveolae. In contrast, cell-bound PDGF-BB delivered via cell-cell contact results in activation and the subsequent up-regulation of a cohort of PDGF beta-receptors primarily confined to the non-lipid raft component of the cell membrane. Individual activation of these two receptor cohorts results in distinct biological endpoints, cytoskeletal reorganization or cell proliferation. Mechanistically, our evidence suggests that PDGF-BB-bearing cells preferentially stimulate the non-lipid raft receptor cohort through interleukin 1beta-mediated inhibition of the lipid raft cohort of receptors, leaving the non-raft receptor cohort operational and preferentially stimulated. In human skin injected with PDGF-BB and in tissue reparative processes PDGF beta-receptors colocalize with the caveolae/lipid raft marker caveolin-1. In contrast, in human skin injected with PDGF-BB-bearing tumor cells and in colorectal adenocarcinoma, activated PDGF beta-receptors do not colocalize with caveolin-1. Thus, growth factor receptors are segregated into specific cell membrane compartments that are preferentially activated through different mechanisms of ligand delivery, resulting in distinct biological endpoints.
Stroma properties affect carcinoma physiology and direct malignant cell development. Here we present data showing that ?(V)?(3) expressed by stromal cells is involved in the control of interstitial fluid pressure (IFP), extracellular volume (ECV) and collagen scaffold architecture in experimental murine carcinoma. IFP was elevated and ECV lowered in syngeneic CT26 colon and LM3 mammary carcinomas grown in integrin ?(3)-deficient compared to wild-type BALB/c mice. Integrin ?(3)-deficiency had no effect on carcinoma growth rate or on vascular morphology and function. Analyses by electron microscopy of carcinomas from integrin ?(3)-deficient mice revealed a coarser and denser collagen network compared to carcinomas in wild-type littermates. Collagen fibers were built from heterogeneous and thicker collagen fibrils in carcinomas from integrin ?(3)-deficient mice. The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin ?(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation. In conclusion, we report that integrin ?(3)-deficiency in tumor stroma led to an elevated IFP and lowered ECV that correlated with a more fibrotic ECM, underlining the role of the collagen network for carcinoma physiology.
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