Writing Committee for the REMAP-CAP Investigators IMPORTANCE The evidence for benefit of convalescent plasma for critically ill patients with COVID-19 is inconclusive.OBJECTIVE To determine whether convalescent plasma would improve outcomes for critically ill adults with COVID-19. DESIGN, SETTING, AND PARTICIPANTSThe ongoing Randomized, Embedded, Multifactorial, Adaptive Platform Trial for Community-Acquired Pneumonia (REMAP-CAP) enrolled and randomized 4763 adults with suspected or confirmed COVID-19 between March 9, 2020, and January 18, 2021, within at least 1 domain; 2011 critically ill adults were randomized to open-label interventions in the immunoglobulin domain at 129 sites in 4 countries. Follow-up ended on April 19, 2021. INTERVENTIONSThe immunoglobulin domain randomized participants to receive 2 units of high-titer, ABO-compatible convalescent plasma (total volume of 550 mL ± 150 mL) within 48 hours of randomization (n = 1084) or no convalescent plasma (n = 916). MAIN OUTCOMES AND MEASURESThe primary ordinal end point was organ support-free days (days alive and free of intensive care unit-based organ support) up to day 21 (range, −1 to 21 days; patients who died were assigned -1 day). The primary analysis was an adjusted bayesian cumulative logistic model. Superiority was defined as the posterior probability of an odds ratio (OR) greater than 1 (threshold for trial conclusion of superiority >99%). Futility was defined as the posterior probability of an OR less than 1.2 (threshold for trial conclusion of futility >95%). An OR greater than 1 represented improved survival, more organ support-free days, or both. The prespecified secondary outcomes included in-hospital survival; 28-day survival; 90-day survival; respiratory support-free days; cardiovascular support-free days; progression to invasive mechanical ventilation, extracorporeal mechanical oxygenation, or death; intensive care unit length of stay; hospital length of stay; World Health Organization ordinal scale score at day 14; venous thromboembolic events at 90 days; and serious adverse events. RESULTS Among the 2011 participants who were randomized (median age, 61 [IQR, 52 to 70] years and 645/1998 [32.3%] women), 1990 (99%) completed the trial. The convalescent plasma intervention was stopped after the prespecified criterion for futility was met. The median number of organ support-free days was 0 (IQR, -1 to 16) in the convalescent plasma group and 3 (IQR, -1 to 16) in the no convalescent plasma group. The in-hospital mortality rate was 37.3% (401/1075) for the convalescent plasma group and 38.4% (347/904) for the no convalescent plasma group and the median number of days alive and free of organ support was 14 (IQR, 3 to 18) and 14 (IQR, 7 to 18), respectively. The median-adjusted OR was 0.97 (95% credible interval, 0.83 to 1.15) and the posterior probability of futility (OR <1.2) was 99.4% for the convalescent plasma group compared with the no convalescent plasma group. The treatment effects were consistent across the primary outcome and the 11...
Abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are hallmark characteristics of vascular remodeling in pulmonary hypertension induced by chronic hypoxia. In this study, we investigated the role of the Na+/H+ exchanger (NHE) and alterations in intracellular pH (pHi) homeostasis in meditating increased proliferation and migration in PASMCs isolated from resistance‐sized pulmonary arteries from chronically hypoxic rats or from normoxic rats that were exposed to hypoxia ex vivo (1% or 4% O2, 24–96 h). We found that PASMCs exposed to either in vivo or ex vivo hypoxia exhibited greater proliferative and migratory capacity, elevated pHi, and enhanced NHE activity. The NHE inhibitor, ethyl isopropyl amiloride (EIPA), normalized pHi in hypoxic PASMCs and reduced migration by 73% and 45% in cells exposed to in vivo and in vitro hypoxia, respectively. Similarly, EIPA reduced proliferation by 97% and 78% in cells exposed to in vivo and in vitro hypoxia, respectively. We previously demonstrated that NHE isoform 1 (NHE1) is the predominant isoform expressed in PASMCs. The development of hypoxia‐induced pulmonary hypertension and alterations in PASMC pH i homeostasis were prevented in mice deficient for NHE1. We found that short‐term (24 h) ex vivo hypoxic exposure did not alter the expression of NHE1, so we tested the role of Rho kinase (ROCK) as a possible means of increasing NHE activity. In the presence of the ROCK inhibitor, Y‐27632, we found that pHi and NHE activity were normalized and migration and proliferation were reduced in PASMCs exposed to either in vivo (by 68% for migration and 22% for proliferation) or ex vivo (by 43% for migration and 17% for proliferation) hypoxia. From these results, we conclude that during hypoxia, activation of ROCK enhances NHE activity and promotes PASMC migration and proliferation.
Excessive production of endothelin‐1 (ET‐1) has been observed in almost all forms of pulmonary hypertension. ET‐1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pH i), typically due to activation of Na+/H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET‐1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET‐1‐induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH‐sensitive dye, BCECF‐AM, to measure changes in resting pH i and NHE activity induced by exposure to exogenous ET‐1 (10−8 mol/L) for 24 h. Cell migration and proliferation in response to ET‐1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET‐1 had no effect on NHE1 expression, but increased pH i, NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET‐1‐induced changes in cell function (proliferation and migration). Our results indicate that ET‐1 modulates PASMC migration and proliferation via changes in pH i homeostasis through a pathway involving ROCK.
Exposure to hypoxia, due to high altitude or chronic lung disease, leads to structural changes in the pulmonary vascular wall, including hyperplasia and migration of pulmonary arterial smooth muscle cells (PASMCs). Previous studies showed that hypoxia upregulates the expression of Na+/H+ exchanger isoform 1 (NHE1) and that inhibition or loss of NHE1 prevents hypoxia-induced PASMC migration and proliferation. The exact mechanism by which NHE1 controls PASMC function has not been fully delineated. In fibroblasts, NHE1 has been shown to act as a membrane anchor for actin filaments, via binding of the adaptor protein, ezrin. Thus, in this study, we tested the role of ezrin and NHE1/actin interactions in controlling PASMC function. Using rat PASMCs exposed to in vitro hypoxia (4% O2, 24 h) we found that hypoxic exposure increased phosphorylation (activation) of ezrin, and promoted interactions between NHE1, phosphorylated ezrin and smooth muscle specific α-actin (SMA) as measured via immunoprecipitation and co-localization. Overexpression of wild-type human NHE1 in the absence of hypoxia was sufficient to induce PASMC migration and proliferation, whereas inhibiting ezrin phosphorylation with NSC668394 suppressed NHE1/SMA co-localization and migration in hypoxic PASMCs. Finally, overexpressing a version of human NHE1 in which amino acids were mutated to prevent NHE1/ezrin/SMA interactions was unable to increase PASMC migration and proliferation despite exhibiting normal Na+/H+ exchange activity. From these results, we conclude that hypoxic exposure increases ezrin phosphorylation in PASMCs, leading to enhanced ezrin/NHE1/SMA interaction. We further speculate that these interactions promote anchoring of the actin cytoskeleton to the membrane to facilitate the changes in cell movement and shape required for migration and proliferation.
Increased PASMC contraction and growth during chronic hypoxia (CH) may be due to cytoskeletal rearrangement involving transmembrane and cytosolic proteins. We found that the ion transporter, NHE1, is upregulated in chronically hypoxic rats (10% O2; 3 wk) and is required for development of hypoxic pulmonary hypertension. In addition to regulating pH, NHE1 was recently found to bind phosphorylated ezrin (p‐ezrin), an actin filament binding protein. NHERF1 also binds p‐ezrin, at the same site as NHE1. Since the transmembrane spanning NHE1 can act as an anchor, but cytosolic NHERF1 cannot, we hypothesized that changes in NHE1/ezrin interactions during hypoxia enables actin filaments to be tethered to the cell membrane, promoting PASMC contraction and growth. Confocal images of immunofluorescent stained chronically hypoxic mouse lung sections confirmed co‐localization of p‐ezrin and smooth muscle cell‐specific α‐actin (SM‐actin). Immunoblots showed increased NHE1, increased p‐ezrin and decreased NHERF1 levels in response to hypoxia. Co‐immunoprecipitation studies showed increased NHE1 binding to p‐ezrin and SM‐actin with hypoxia (4% O2; 24 hr), and decreased NHERF1 binding. These results suggest reciprocal regulation of NHERF1 and NHE1 expression in PASMC during hypoxia, with both proteins competing to bind p‐ezrin. Increased NHE1/ezrin interactions may contribute to hypoxic pulmonary hypertension.
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