We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity.

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.

The intermediate filament framework is one of the three cytoskeletal systems in mammalian cells. Its well spread filamentous structure from the nucleus to the plasma membrane is believed to provide protection against various mechanical stresses (1, 2). Intermediate filaments also undergo disassembly/assembly and spatial reorganization in cells in response to external stimulation and during mitosis. The dynamic property of the intermediate filament system plays a fundamental role in mediating changes in cell shape, division, and migration; signaling molecule distribution; and smooth muscle force development (1, 3-7).The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation. Vimentin is the most abundant intermediate filament protein in various cell types, including smooth muscle cells (2,5,6). Vimentin phosphorylation in association with vimentin disassembly and spatial reorganization occurs during mitosis or in response to extracellular stimulation (8, 9). In cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation at Ser-56 concurrently with vimentin partial disassembly and spatial reorientation (6).The disassembly and spatial reorganization of the vimentin network may regulate the translocation of certain molecules (7, 10, 11). The adapter protein p130 Crk-associated substrate (CAS) 2 has been shown to participate in the signaling processes that regulate smooth muscle contraction and cell migration (12-14). Our recent study has suggested that vimentin phosphorylation and disassembly are related to CAS redistribution during contractile activation of smooth muscle (10). In addition, external stress initiates Rho kinase redistribution associated with vimentin depolymerization in fibroblasts and the translocation of Ca 2ϩ /calmodulin-dependent protein kinase II in differentiated smooth muscle cells, which may be an important event for cell signaling (7, 11).p21-activated kinase (PAK) may be an upstream regulator of the vimentin network (6, 9). In cultured smooth muscle cells, agonist-mediated vimentin phosphorylation at Ser-56 and spatial reorientation of the vimentin network are inhibited by silencing of PAK1, a dominant isoform in smooth muscle (6,15). Additionally, PAK has been implicated in modulating smooth muscle contraction; introduction of an active PAK isoform into smooth muscle potentiates force development at constant intracellular calcium (16). Expression of an inactive PAK1 mutant attenuates migration of cultured smooth muscle cells in response to platelet-derived growth factor (15).In response to external stimulation, PAK undergoes autophosphorylation at Thr-423, which increases PAK activity for substrates (17,18). In addition to the small GTPases Cdc42 and Rac1, the activity of PAK may be regulated by the paxillin kinase linker/PIX (PAK-interacting exchange factor; guanine nucleotide exchange factor) (19 -21). CAS has been shown to interact with the paxillin kinase linker/PIX via CrkII and paxillin (21-* This work was ...

SUMMARY Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary HCs and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis.

Calcium (Ca 2+ ) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca 2+ permeable channels, Ca 2+ pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca 2+ levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca 2+ signaling pathways that regulate VSMC contraction are the plasma membrane voltageoperated Ca 2+ channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca 2+ release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca 2+ signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca 2+ channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca 2+ release channels, pumps and Ca 2+ -activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase isoform 2a pump and reciprocally regulate isoforms of the ca 2+ /calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca 2+ signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca 2+ signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.