Natalia I. Dmitrieva

Helper B Cells Promote Cytotoxic T Cell Survival and Proliferation Independently of Antigen Presentation through CD27/CD70 Interactions

CD8-expressing cytotoxic T cell (CTL) interactions with APCs and helper T cells determine their function and ability to survive. In this study, we describe a novel interaction independent of Ag presentation between activated CTLs and bystander CD19-expressing B lymphocytes. Ag-stimulated CTLs serially engage autologous B lymphocytes through CD27/CD70 contact that promotes their survival and proliferation. Moreover, these interactions induce the release of proinflammatory cytokines that follows two general patterns: 1) an epitope-dependent enhancement of cytokine release, and 2) a previously undiscovered coordinate release of cytokines independent of epitope exposure. The latter includes chemoattractants targeting activated T cells. As a result, activated T cells are attracted to B cells, which exert a “helper” role in lymphatic organs or in areas of inflammation. This observation provides a mechanistic explanation to previously reported experimental observations suggesting that B cells are required for T cell priming in vivo.

Secretion of von Willebrand factor by endothelial cells links sodium to hypercoagulability and thrombosis

Significance Cardiovascular thrombotic events are a leading cause of mortality and morbidity worldwide. Hypercoagulability increases the risk of thrombosis. This study shows that elevated sodium concentration stimulates endothelial production of a key initiator of the clotting cascade, von Willebrand factor, leading to increased coagulation and thrombogenesis. In everyday life, increases in blood sodium often occur as the result of insufficient drinking, excessive water loss, or high salt intake. Therefore, our results indicate that water and salt intake are modifiable factors affecting coagulability and risk of thrombosis. In clinical practice, sustained elevation of plasma sodium is a part of widely used hypertonic saline therapy. Our results suggest that monitoring of coagulation during the therapy might be beneficial to prevent thrombotic complications. Hypercoagulability increases risk of thrombi that cause cardiovascular events. Here we identify plasma sodium concentration as a factor that modulates blood coagulability by affecting the production of von Willebrand factor (vWF), a key initiator of the clotting cascade. We find that elevation of salt over a range from the lower end of what is normal in blood to the level of severe hypernatremia reversibly increases vWF mRNA in endothelial cells in culture and the rate of vWF secretion from them. The high NaCl increases expression of tonicity-regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting involvement of hypertonic signaling in vWF up-regulation. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) by feeding them with gel food containing 30% water. Such WR elevates blood sodium from 145.1 ± 0.5 to 150.2 ± 1.3 mmol/L and activates hypertonic signaling, evidenced from increased expression of NFAT5 in tissues. WR increases vWF mRNA in liver and lung and raises vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increases blood level of D-dimer, indicative of ongoing coagulation and thrombolysis. Multivariate regression analysis of clinical data from the Atherosclerosis Risk in Communities Study demonstrated that serum sodium significantly contributes to prediction of plasma vWF and risk of stroke. The results indicate that elevation of extracellular sodium within the physiological range raises vWF sufficiently to increase coagulability and risk of thrombosis.

Living with DNA breaks is an everyday reality for cells adapted to high NaCl.

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.

Osmotic stress and DNA damage.

Mammalian renal inner medullary cells are normally exposed to extremely high NaCl concentrations. The interstitial NaCl concentration in parts of a normal renal medulla can be 500 mM or more, depending on the species. Remarkably, under these normal conditions, the high NaCl causes DNA damage, yet the cells survive and function both in cell culture and in vivo. Both in cell culture and in vivo the breaks are repaired rapidly if the NaCl concentration is lowered. This chapter describes two methods used to detect and study the DNA damage induced by osmotic stress: comet assay or single cell electrophoresis and TUNEL assay or in situ labeling of 3-OH ends of DNA strands. This chapter also discusses how specifics of the protocols influence the conclusions about types of DNA damage and what the limitations of these methods are for detecting different types of DNA damage.

Elevated Sodium and Dehydration Stimulate Inflammatory Signaling in Endothelial Cells and Promote Atherosclerosis

Cardiovascular diseases (CVDs) are a leading health problem worldwide. Epidemiologic studies link high salt intake and conditions predisposing to dehydration such as low water intake, diabetes and old age to increased risk of CVD. Previously, we demonstrated that elevation of extracellular sodium, which is a common consequence of these conditions, stimulates production by endothelial cells of clotting initiator, von Willebrand Factor, increases its level in blood and promotes thrombogenesis. In present study, by PCR array, using human umbilical vein endothelial cells (HUVECs), we analyzed the effect of high NaCl on 84 genes related to endothelial cell biology. The analysis showed that the affected genes regulate many aspects of endothelial cell biology including cell adhesion, proliferation, leukocyte and lymphocyte activation, coagulation, angiogenesis and inflammatory response. The genes whose expression increased the most were adhesion molecules VCAM1 and E-selectin and the chemoattractant MCP-1. These are key participants in the leukocyte adhesion and transmigration that play a major role in the inflammation and pathophysiology of CVD, including atherosclerosis. Indeed, high NaCl increased adhesion of mononuclear cells and their transmigration through HUVECs monolayers. In mice, mild water restriction that elevates serum sodium by 5 mmol/l, increased VCAM1, E-selectin and MCP-1 expression in mouse tissues, accelerated atherosclerotic plaque formation in aortic root and caused thickening or walls of coronary arteries. Multivariable linear regression analysis of clinical data from the Atherosclerosis Risk in Communities Study (n=12779) demonstrated that serum sodium is a significant predictor of 10 Years Risk of coronary heart disease. These findings indicate that elevation of extracellular sodium within the physiological range is accompanied by vascular changes that facilitate development of CVD. The findings bring attention to serum sodium as a risk factor for CVDs and give additional support to recommendations for dietary salt restriction and adequate water intake as preventives of CVD.

Increased insensible water loss contributes to aging related dehydration.

Dehydration with aging is attributed to decreased urine concentrating ability and thirst. We further investigated by comparing urine concentration and water balance in 3, 18 and 27 month old mice, consuming equal amounts of water. During water restriction, 3 month old mice concentrate their urine sufficiently to maintain water balance (stable weight). 18 month old mice concentrate their urine as well, but still lose weight (negative water balance). 27 month old mice do not concentrate their urine as well and lose even more weight than the 18 month old mice, indicating a larger negative water balance. Negative water balance in older mice is accompanied by increased vasopressin excretion, providing further evidence of dehydration. All 3 groups maintain water balance while consuming only the water in gel food containing 56% water. However, both older groups excrete a smaller volume of urine of higher osmolality, indicating greater extra urinary water loss. Since their feces also contain less water, the excess water lost by the older mice apparently is through other routes, presumably insensible loss through the respiratory tract and skin. The greater insensible water loss occurs at an earlier age (18 months) than decreased urine concentrating ability (27 months). We propose that insensible water loss through skin and respiration increases with age, making a major contribution to aging related dehydration.

Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC

Patient-derived induced pluripotent stem cells reveal treatment strategies for a rare genetic form of arterial calcification. Understanding vascular calcification ACDC is a rare genetic vascular calcification disease caused by loss of CD73, a secreted enzyme that converts adenosine monophosphate (AMP) to adenosine. Cells from ACDC patients have a compensatory increase in the phosphatase TNAP, which primarily catalyzes the conversion of pyrophosphate to inorganic phosphate but can also convert AMP to adenosine. Jin et al. generated induced pluripotent stem cells (iPSCs) from ACDC patients. Although in culture, these cells generated adenosine from AMP, the cells had decreased amounts of pyrophosphate, which inhibits calcification. ACDC patient–derived cells showed increased activation of the mTOR pathway, which promotes calcification. When injected into mice, the ACDC patient–derived iPSCs formed calcified teratomas. Treating mice bearing these teratomas with an adenosine receptor agonist, the mTOR inhibitor rapamycin, or etidronate (a drug that is structurally similar to pyrophosphate) reduced calcification in the teratomas, suggesting multiple potential strategies for treating ectopic calcification in ACDC patients and thereby alleviating the pain and peripheral ischemia associated with the disease. ACDC (arterial calcification due to deficiency of CD73) is an autosomal recessive disease resulting from loss-of-function mutations in NT5E, which encodes CD73, a 5′-ectonucleotidase that converts extracellular adenosine monophosphate to adenosine. ACDC patients display progressive calcification of lower extremity arteries, causing limb ischemia. Tissue-nonspecific alkaline phosphatase (TNAP), which converts pyrophosphate (PPi) to inorganic phosphate (Pi), and extracellular purine metabolism play important roles in other inherited forms of vascular calcification. Compared to cells from healthy subjects, induced pluripotent stem cell–derived mesenchymal stromal cells (iMSCs) from ACDC patients displayed accelerated calcification and increased TNAP activity when cultured under conditions that promote osteogenesis. TNAP activity generated adenosine in iMSCs derived from ACDC patients but not in iMSCs from control subjects, which have CD73. In response to osteogenic stimulation, ACDC patient–derived iMSCs had decreased amounts of the TNAP substrate PPi, an inhibitor of extracellular matrix calcification, and exhibited increased activation of AKT, mechanistic target of rapamycin (mTOR), and the 70-kDa ribosomal protein S6 kinase (p70S6K), a pathway that promotes calcification. In vivo, teratomas derived from ACDC patient cells showed extensive calcification and increased TNAP activity. Treating mice bearing these teratomas with an A2b adenosine receptor agonist, the mTOR inhibitor rapamycin, or the bisphosphonate etidronate reduced calcification. These results show that an increase of TNAP activity in ACDC contributes to ectopic calcification by disrupting the extracellular balance of PPi and Pi and identify potential therapeutic targets for ACDC.

Global discovery of high-NaCl-induced changes of protein phosphorylation

High extracellular NaCl, such as in the renal medulla, can perturb and even kill cells, but cells mount protective responses that enable them to survive and function. Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyze the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death.