Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis.
Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA-approved drug library.
Aims: Macropinocytosis has been implicated in cardiovascular and other disorders, yet physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved. Results: TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and NADPH oxidase 1 (Nox1) signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin-related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser-3, actin remodeling, and macropinocytotic uptake of unmodified native low-density lipoprotein (nLDL), leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE−/− mice in vivo. Innovation: Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to early propagation of vascular inflammatory disease. Conclusions: The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated. Antioxid. Redox Signal. 26, 886–901.
The pyruvate dehydrogenase complex (PDC) plays a critical role in lipid synthesis and glucose homeostasis in the fed and fasting states. The central role of the liver in the maintenance of glucose homeostasis has been established by studying changes in key enzymes (including PDC) and the carbon-flux via several pathways under different metabolic states. In the present study we have developed a murine model of liver-specific PDC deficiency using Cre-loxP technology to investigate its consequences on lipid and carbohydrate metabolism. There was no incorporation of glucose-carbon into fatty acids by liver in vitro from liver-specific Pdha1 knockout (L-PDHKO) male mice due to absence of hepatic PDC activity. Interestingly, there was a compensatory increase in lipogenic capacity in epididymal adipose tissue from L-PDHKO mice. Both fat and lean body mass were significantly reduced in L-PDHKO mice, which might be explained by an increase in total energy expenditure compared with wild-type littermate mice. Furthermore, both liver and peripheral insulin sensitivities measured during a hyperinsulinemic-euglycemic clamp were improved in L-PDHKO mice. The findings presented here demonstrate (i) the indispensable role of PDC for lipogenesis from glucose in liver and (ii) specific adaptations in lipid and glucose metabolism in the liver and adipose tissue to compensate for loss of PDC activity in liver only.
Internalization of extracellular fluid and its solute by macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase C (PKC), are involved in macropinocytosis, the role of Nox enzymes in macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in macropinocytosis.Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and scavenging of intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. We observed that Nox2-derived reactive oxygen species via inhibition of phosphatase and tensin homolog and activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway lead to activation of actin-binding protein cofilin, membrane ruffling, and macropinocytosis. Similarly, activation of macropinocytosis by macrophage colony-stimulating factor involves Nox2-mediated cofilin activation. Furthermore, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE-/- mice was attenuated in Nox2y/- macrophages compared with wild-type controls. Innovation and Conclusion: In summary, these findings demonstrate a novel Nox2-mediated mechanism of solute uptake via macropinocytosis, with broad implications for both general cellular physiology and pathological processes. The redox mechanism described here may also identify new targets in atherosclerosis and other disease conditions involving macropinocytosis. Antioxid. Redox Signal. 26, 902-916.
Abstract Indolent non-Hodgkin lymphomas are characterized by a prolonged phase that is typically followed by a clinical progression associated with an accelerated clinical course and short survival time. Previous studies have not identified a consistent cytogenetic or molecular abnormality associated with transformation. The development of a transformed phenotype, evolving from the original low-grade component, most likely depends on multiple genetic events, including the activation of synergistic dominant oncogenes and a loss of tumor suppressor gene functions. Complex karyotypes and relatively bad chromosome morphology are typical of transformed non-Hodgkin lymphomas, rendering complete cytogenetic analysis difficult. Here, we report the use of transformed non-Hodgkin lymphoma cell lines and primary samples to identify the involvement of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) gene that maps at chromosome 12q24 in transformed non-Hodgkin lymphomas. We also show that down-regulation of SMRT in the immortalized “Weinberg's model” cell lines induces transformation of the cells. Assessment of cDNA array profiles should further help us to design a working model for SMRT involvement in non-Hodgkin lymphoma transformation as a novel, nonclassical tumor suppressor.
Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.
