Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)[superscript +] macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE[superscript −/−] mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes

Abstract Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes

Protective effects of the mTOR inhibitor everolimus on cytoskeletal injury in human podocytes are mediated by RhoA signaling

Podocytes are highly differentiated kidney cells playing an important role in maintaining the glomerular filtration barrier. Particularly, the integrity of the actin cytoskeleton is crucial as cytoskeletal damage associated with foot process effacement and loss of slit diaphragms constitutes a major aspect of proteinuria. Previously, the mammalian target of rapamycin (mTOR) was linked to actin regulation and aberrant activity of the kinase was associated with renal disease. In this study, actin-related effects of mTOR inhibition by the immunosuppressant everolimus (EV) were investigated in human podocytes using an in vitro model of puromycin aminonucleoside (PAN) induced proteinuria. EV substantially recovered aberrant podocyte behavior by re-establishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers. Biochemical studies revealed substantial increase in the activity of RhoA and the effector pathway Rho-associated protein kinase (ROCK) and myosin light chain (MLC) by EV, all known regulators of stress fiber generation. Taken together, we show for the first time cytoskeleton stabilizing effects of the mTOR inhibitor EV and establish RhoA signaling as a key mediator in this process

Angiotensin-II-receptor inhibitors in pregnancy [3]

SCOPUS: le.jinfo:eu-repo/semantics/publishe

EV inhibits mTORC1 and mTORC2.

<p>(A) Western blot analysis to measure the activation level of Akt (representative example from 3 independent experiments). GAPDH = loading control, Akt = total Akt protein levels, pAkt = active, phosphorylated Akt protein. (B) For quantification, total Akt protein was normalized with respect to GAPDH. (C) To quantify the amount of active Akt protein, phosphorylated Akt was normalized with respect to total Akt. (D) Western blot analysis of p70S6K protein (representative example from 3 independent experiments). GAPDH = loading control, p70S6K = total p70S6K protein levels, p-p70S6K = active, phosphorylated p70S6K protein. (E) For quantification, total p70S6K was normalized to GAPDH. (F) Phosphorylated p70S6K was normalized to total p70S6K.</p

EV targets RhoA signaling pathway in human podocytes.

<p>(A) Biochemical assay to measure the activation level of the GTPase. The Rho-binding domain (RBD) of the RhoA effector rhotekin was used to affinity-precipitate the active fraction of endogenous RhoA (GTP-RhoA) from cell lysates (representative example from 3 independent experiments). Tubulin was used as loading control. (B) Quantification of total RhoA protein (n = 3 experiments). For quantification, total RhoA protein was normalized with respect to tubulin from whole cell lysates. (C) To quantify the amount of active RhoA protein, GTP-bound RhoA was normalized with respect to total RhoA (n = 3 experiments). (D) Western-blot analysis of MLC protein (representative example from 4 independent experiments). GAPDH = loading control, MLC = total MLC protein levels, pMLC = active, phosphorylated MLC protein. (E) Quantification of total MLC protein (n = 4 experiments). For quantification, total MLC was normalized to GAPDH from whole cell lysates. (F) Quantification of phosphorylated MLC protein (n = 4 experiments). Phosphorylated MLC was normalized to total MLC from whole cell lysates. (G) Western blot analysis of MLC protein after treatment with the ROCK inhibitor Y-27632 (10 µM for 1 h; n = 2 independent experiments). (H) Actin cytoskeleton (phalloidin-TRITC, grey) after treatment with Y-27632. DAPI was used for nuclear staining (blue). Scale bar = 100 µm. MeOH = solvent for EV. Data are means ± SD.</p

EV inhibits migration in human podocytes.

<p>(A) Representative phase contrast images after 0 h and 12 h of migration. Scale bar = 500 µm. (B) Quantification of migration efficiency by measurement of cell-free area after 12 h of migration (n = 3 experiments, ≥5 images per condition). (C) Phase contrast time-lapse studies of living cells. Migration was tracked following the nuclei in the phase contrast movie. Lower panel: Tracks were depicted on white background for better contrast. Scale bar = 500 µm. MeOH = solvent for EV. Data are means ± SD.</p