No abstract

The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently become a topic of intense scrutiny. Here, we characterized the behavior of MC/Mph in cellular infiltrates, with emphasis on their spatial organization and localization in newly formed microvessels. To this end, we studied MC/Mph migration and assembly in basic fibroblast growth factor-supplemented Matrigel plugs placed in transgenic Tie2-beta-galactosidase mice for up to 4 weeks. In these plugs, along with Nile Red-positive adipocytes, we found MC/Mph distributed in cell cords, also containing various mature and progenitor tissue cells; and functional Tie2-positive or -negative microvessels embedded in bundles of fibrillar collagen surrounded by F4/80-positive MC/Mph. At earlier stages of infiltration, we found tubular destruction of the matrix (tunnels) and MC/Mph-lined capillary-like structures occasionally containing erythrocytes, indicating their propensity for endothelial trans-differentiation. We also analyzed in vitro the MCP-1-induced chemotactic migration of fluorescently labeled peritoneal MC/Mph incorporated in Matrigel-containing fluorescent protease substrates. Many of these MC/Mph produced MMP-12- and TIMP-1-dependent tunnels coupled with acquisition of a lumen. In conclusion, long-term implantation of Matrigel plugs qualifies as a novel experimental model of tissue regeneration, in which neovascularization intimately couples with fibrosis and organogenesis and in which cells of MC/Mph phenotype play a key structural role.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

Newly emerging Omicron subvariants continue to emerge around the world, presenting potential challenges to current vaccination strategies. This study investigates the extent of neutralizing antibody escape by new subvariants XBB.1.5, CH.1.1, and CA.3.1, as well as their impacts on spike protein biology. Our results demonstrated a nearly complete escape of these variants from neutralizing antibodies stimulated by three doses of mRNA vaccine, but neutralization was rescued by a bivalent booster. However, CH.1.1 and CA.3.1 variants were highly resistant to both monovalent and bivalent mRNA vaccinations. We also assessed neutralization by sera from individuals infected during the BA.4/5 wave of infection and observed similar trends of immune escape. In these cohorts, XBB.1.5 did not exhibit enhanced neutralization resistance over the recently dominant BQ.1.1 variant. Notably, the spike proteins of XBB.1.5, CH.1.1, and CA.3.1 all exhibited increased fusogenicity compared to BA.2, correlating with enhanced S processing. Overall, our results support the administration of new bivalent mRNA vaccines, especially in fighting against newly emerged Omicron subvariants, as well as the need for continued surveillance of Omicron subvariants.

Continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants including BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2. Here we examine the neutralization resistance of these subvariants as well as their ancestral BA.4/5, BA.2.75 and D614G variants against sera from 3-dose vaccinated health care workers, hospitalized BA.1-wave patients, and BA.5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially the BQ.1 and BQ.1.1 subvariants driven by a key N460K mutation and to a lesser extent, R346T and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its key F486S mutation. The BQ.1 and BQ.1.1 subvariants also exhibited enhanced fusogenicity and S processing dictated by the key N460K mutation. Interestingly, the BA.2.75.2 subvariant saw an enhancement by the F486S mutation and a reduction by the D1199N mutation to its fusogenicity and S processing resulting in minimal overall change. Molecular modelling revealed the mechanisms of receptor-binding and non-receptor binding monoclonal antibody-mediated immune evasion by R346T, K444T, F486S and D1199N mutations. Altogether, these findings shed light on the concerning evolution of newly emerging SARS-CoV-2 Omicron subvariants.

Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to neovascularization, but their nature and significance remains elusive. Based on the emerging evidence implicating a role for monocytes and macrophages (MC/MPH) in vasculogenesis, we hypothesized that MC/MPH also can form linear or branched columns, facilitating the co-migration and the spatial arrangement of other cell types. To test this hypothesis, we studied the distribution of MC/MPH effected by chemotactic migration in novel in vitro and in vivo models of development. We induced transversal and lateral migration of THP-1 monocytoid cells in Matrigel in vitro. The effect of this process on co-localization of other micro-objects was assessed using erythrocytes and micron-sized plastic beads. In vivo, we analyzed MC/MPH infiltration in subcutaneously implanted Matrigel plugs containing angiogenic factors and across a microporous filter comprising the wall of a chamber filled with Matrigel, also placed subcutaneously in mice. In vitro, we found that migrating THP-1 cells induced the lasting degradation of Matrigel and produced cell columns, a process amplified by monocyte chemoattractant protein-1 (MCP-1). We also report the co-localization of erythrocytes with THP-1 cells in cell columns. Endothelium-free tunnels containing MC/MPH, neutrophils, or erythrocytes were also observed in the Matrigel-filled chambers. In free subcutaneous Matrigel plugs, we found MC/MPH-based columns harboring isolated Tie-2+ cells (a marker of endothelial progenitor phenotype), as well as fibroblasts, dendritic cells, and adypocytes. Many of these cell columns displayed conspicuous branching. Our data demonstrate formation of branched MC/MPH cell columns in vitro and in vivo, a previously unrecognized pattern of penetration of extracellular matrices by inflammatory cells. Thus, monocytes and macrophages influence the distribution of neovessels as well as their branching points. These cells are the "architects of development," assisting organogenesis, tumorigenesis, and wound healing by patterning the tissular space.

No abstract

Objective. While the effects of biomechanical signals in the form of joint movement and exercise are known to be beneficial to inflamed joints, limited information is available regarding the intracellular mechanisms of their actions. This study was undertaken to examine the intracellular mechanisms by which biomechanical signals suppress proinflammatory gene induction by the interleukin-1-␤ (IL-1␤)-induced NF-B signaling cascade in articular chondrocytes.Methods. Primary rat articular chondrocytes were exposed to biomechanical signals in the form of cyclic tensile strain, and the effects on the NF-B signaling cascade were examined by Western blot analysis, real-time polymerase chain reaction, and immunofluorescence.Results. Cyclic tensile strain rapidly inhibited the IL-1␤-induced nuclear translocation of NF-B, but not its IL-1␤-induced phosphorylation at serine 276 and serine 536, which are necessary for its transactivation and transcriptional efficacy, respectively. Examination of upstream events revealed that cyclic tensile strain also inhibited the cytoplasmic protein degradation of IB␤ and IB␣, as well as repressed their gene transcription. Additionally, cyclic tensile strain induced a rapid nuclear translocation of IB␣ to potentially prevent NF-B binding to DNA. Furthermore, the inhibition of IL-1␤-induced degradation of IB by cyclic tensile strain was mediated by down-regulation of IB kinase activity.Conclusion. These results indicate that the signals generated by cyclic tensile strain act at multiple sites within the NF-B signaling cascade to inhibit IL-1␤-induced proinflammatory gene induction. Taken together, these findings provide insight into how biomechanical signals regulate and reduce inflammation, and underscore their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints.