Tackling Brain and Muscle Dysfunction in Acute Respiratory Distress Syndrome Survivors: NHLBI Workshop Report.

Acute respiratory distress syndrome (ARDS) is associated with long-term impairments in brain and muscle function that significantly impact the quality of life of those who survive the acute illness. The mechanisms underlying these impairments are not yet well understood, and evidence-based interventions to minimize the burden on patients remain unproved. The NHLBI of the NIH assembled a workshop in April 2023 to review the state of the science regarding ARDS-associated brain and muscle dysfunction, to identify gaps in current knowledge, and to determine priorities for future investigation. The workshop included presentations by scientific leaders across the translational science spectrum and was open to the public as well as the scientific community. This report describes the themes discussed at the workshop as well as recommendations to advance the field toward the goal of improving the health and well-being of ARDS survivors.

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes. METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay. RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05). CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

Preclinical and translational models for delirium: Recommendations for future research from the NIDUS delirium network.

Delirium is a common, morbid, and costly syndrome that is closely linked to Alzheimer's disease (AD) and AD-related dementias (ADRD) as a risk factor and outcome. Human studies of delirium have advanced our knowledge of delirium incidence and prevalence, risk factors, biomarkers, outcomes, prevention, and management. However, understanding of delirium neurobiology remains limited. Preclinical and translational models for delirium, while challenging to develop, could advance our knowledge of delirium neurobiology and inform the development of new prevention and treatment approaches. We discuss the use of preclinical and translational animal models in delirium, focusing on (1) a review of current animal models, (2) challenges and strategies for replicating elements of human delirium in animals, and (3) the utility of biofluid, neurophysiology, and neuroimaging translational markers in animals. We conclude with recommendations for the development and validation of preclinical and translational models for delirium, with the goal of advancing awareness in this important field.

Proteoglycans and glycosaminoglycans in central nervous system injury.

The brain and spinal cord constitute the central nervous system (CNS), which when injured, can be exceedingly devastating. The mechanistic roles of proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains in such injuries have been extensively studied. CNS injury immediately alters endothelial and extracellular matrix (ECM) PGs and GAGs. Subsequently, these alterations contribute to acute injury, postinjury fibrosis, and postinjury repair. These effects are central to the pathophysiology of CNS injury. This review focuses on the importance of PGs and GAGs in multiple forms of injury including traumatic brain injury, spinal cord injury, and stroke. We highlight the causes and consequences of degradation of the PG and GAG-enriched endothelial glycocalyx in early injury and discuss the pleiotropic roles of PGs in neuroinflammation. We subsequently evaluate the dualistic effects of PGs on recovery: both PG/GAG-mediated inhibition and facilitation of repair. We then report promising therapeutic strategies that may prove effective for repair of CNS injury including PG receptor inhibition, delivery of endogenous, pro-repair PGs and GAGs, and direct degradation of pathological GAGs. Finally, we discuss the importance of two PG- and GAG-containing ECM structures (synapses and perineuronal nets) in CNS injury and recovery.

Circulating heparin oligosaccharides rapidly target the hippocampus in sepsis, potentially impacting cognitive functions.

Sepsis induces heparanase-mediated degradation of the endothelial glycocalyx, a heparan sulfate-enriched endovascular layer critical to vascular homeostasis, releasing highly sulfated domains of heparan sulfate into the circulation. These domains are oligosaccharides rich in heparin-like trisulfated disaccharide repeating units. Using a chemoenzymatic approach, an undecasaccharide containing a uniformly 13C-labeled internal 2-sulfoiduronic acid residue was synthesized on a p-nitrophenylglucuronide acceptor. Selective periodate cleavage afforded a heparin nonasaccharide having a natural structure. This 13C-labeled nonasaccharide was intravenously administered to septic (induced by cecal ligation and puncture, a model of polymicrobial peritonitis-induced sepsis) and nonseptic (sham) mice. Selected tissues and biological fluids from the mice were harvested at various time points over 4 hours, and the 13C-labeled nonasaccharide was recovered and digested with heparin lyases. The resulting 13C-labeled trisulfated disaccharide was quantified, without interference from endogenous mouse heparan sulfate/heparin, using liquid chromatography-mass spectrometry with sensitive and selective multiple reaction monitoring. The 13C-labeled heparin nonasaccharide appeared immediately in the blood and was rapidly cleared through the urine. Plasma nonasaccharide clearance was only slightly prolonged in septic mice (t1/2 ∼ 90 minutes). In septic mice, the nonasaccharide penetrated into the hippocampus but not the cortex of the brain; no hippocampal or cortical brain penetration occurred in sham mice. The results of this study suggest that circulating heparan sulfates are rapidly cleared from the plasma during sepsis and selectively penetrate the hippocampus, where they may have functional consequences.

Staphylococcus aureus adhesion in endovascular infections is controlled by the ArlRS-MgrA signaling cascade.

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.

Heparin as a therapy for COVID-19: current evidence and future possibilities.

Coronavirus disease 2019 (COVID-19), the clinical syndrome associated with infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted nearly every country in the world. Despite an unprecedented focus of scientific investigation, there is a paucity of evidence-based pharmacotherapies against this disease. Because of this lack of data-driven treatment strategies, broad variations in practice patterns have emerged. Observed hypercoagulability in patients with COVID-19 has created debate within the critical care community on the therapeutic utility of heparin. We seek to provide an overview of the data supporting the therapeutic use of heparin, both unfractionated and low molecular weight, as an anticoagulant for the treatment of SARS-CoV-2 infection. Additionally, we review preclinical evidence establishing biological plausibility for heparin and synthetic heparin-like drugs as therapies for COVID-19 through antiviral and anti-inflammatory effects. Finally, we discuss known adverse effects and theoretical off-target effects that may temper enthusiasm for the adoption of heparin as a therapy in COVID-19 without confirmatory prospective randomized controlled trials. Despite previous failures of anticoagulants in critical illness, plausibility of heparin for COVID-19 is sufficiently robust to justify urgent randomized controlled trials to determine the safety and effectiveness of this therapy.

Intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation.

BACKGROUND: Intravenous fluids, an essential component of sepsis resuscitation, may paradoxically worsen outcomes by exacerbating endothelial injury. Preclinical models suggest that fluid resuscitation degrades the endothelial glycocalyx, a heparan sulfate-enriched structure necessary for vascular homeostasis. We hypothesized that endothelial glycocalyx degradation is associated with the volume of intravenous fluids administered during early sepsis resuscitation. METHODS: We used mass spectrometry to measure plasma heparan sulfate (a highly sensitive and specific index of systemic endothelial glycocalyx degradation) after 6 h of intravenous fluids in 56 septic shock patients, at presentation and after 24 h of intravenous fluids in 100 sepsis patients, and in two groups of non-infected patients. We compared plasma heparan sulfate concentrations between sepsis and non-sepsis patients, as well as between sepsis survivors and sepsis non-survivors. We used multivariable linear regression to model the association between volume of intravenous fluids and changes in plasma heparan sulfate. RESULTS: Consistent with previous studies, median plasma heparan sulfate was elevated in septic shock patients (118 [IQR, 113-341] ng/ml 6 h after presentation) compared to non-infected controls (61 [45-79] ng/ml), as well as in a second cohort of sepsis patients (283 [155-584] ng/ml) at emergency department presentation) compared to controls (177 [144-262] ng/ml). In the larger sepsis cohort, heparan sulfate predicted in-hospital mortality. In both cohorts, multivariable linear regression adjusting for age and severity of illness demonstrated a significant association between volume of intravenous fluids administered during resuscitation and plasma heparan sulfate. In the second cohort, independent of disease severity and age, each 1 l of intravenous fluids administered was associated with a 200 ng/ml increase in circulating heparan sulfate (p = 0.006) at 24 h after enrollment. CONCLUSIONS: Glycocalyx degradation occurs in sepsis and septic shock and is associated with in-hospital mortality. The volume of intravenous fluids administered during sepsis resuscitation is independently associated with the degree of glycocalyx degradation. These findings suggest a potential mechanism by which intravenous fluid resuscitation strategies may induce iatrogenic endothelial injury.

Inflammation, endothelial injury, and the acute respiratory distress syndrome after out-of-hospital cardiac arrest.

Background: Acute respiratory distress syndrome (ARDS) is often seen in patients resuscitated from out-of-hospital cardiac arrest (OHCA). We aim to test whether inflammatory or endothelial injury markers are associated with the development of ARDS in patients hospitalized after OHCA. Methods: We conducted a prospective, cohort, pilot study at an urban academic medical center in 2019 that included a convenience sample of adults with non-traumatic OHCA. Blood and pulmonary edema fluid (PEF) were collected within 12 hours of hospital arrival. Samples were assayed for cytokines (interleukin [IL]-1, tumor necrosis factor-α [TNF-α], tumor necrosis factor receptor1 [TNFR1], IL-6), epithelial injury markers (pulmonary surfactant-associated protein D), endothelial injury markers (Angiopoietin-2 [Ang-2] and glycocalyx degradation products), and other proteins (matrix metallopeptidase-9 and myeloperoxidase). Patients were followed for 7 days for development of ARDS, as adjudicated by 3 blinded reviewers, and through hospital discharge for mortality and neurological outcome. We examined associations between biomarker concentrations and ARDS, hospital mortality, and neurological outcome using multivariable logistic regression. Latent phase analysis was used to identify distinct biological classes associated with outcomes. Results: 41 patients were enrolled. Mean age was 58 years, 29% were female, and 22% had a respiratory etiology for cardiac arrest. Seven patients (17%) developed ARDS within 7 days. There were no significant associations between individual biomarkers and development of ARDS in adjusted analyses, nor survival or neurologic status after adjusting for use of targeted temperature management (TTM) and initial cardiac arrest rhythm. Elevated Ang-2 and TNFR-1 were associated with decreased survival (RR = 0.6, 95% CI = 0.3-1.0; RR = 0.5, 95% CI = 0.3-0.9; respectively), and poor neurologic status at discharge (RR = 0.4, 95% CI = 0.2-0.8; RR = 0.4, 95% CI = 0.2-0.9) in unadjusted associations. Conclusion: OHCA patients have markedly elevated plasma and pulmonary edema fluid biomarker concentrations, indicating widespread inflammation, epithelial injury, and endothelial activation. Biomarker concentrations were not associated with ARDS development, though several distinct biological phenotypes warrant further exploration. Latent phase analysis demonstrated that patients with low biomarker levels aside from TNF-α and TNFR-1 (Class 2) fared worse than other patients. Future research may benefit from considering other tools to predict and prevent development of ARDS in this population.

A role for decorin in improving motor deficits after traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability due to injury worldwide. Extracellular matrix (ECM) remodeling is known to significantly contribute to TBI pathophysiology. Glycosaminoglycans, which are long-chain, variably sulfated polysaccharides abundant within the ECM, have previously been shown to be substantially altered after TBI. In this study, we sought to delineate the dynamics of glycosaminoglycan alterations after TBI and discover the precise biologic processes responsible for observed glycosaminoglycan changes after injury. We performed state-of-the art mass spectrometry on brain tissues isolated from mice after TBI or craniotomy-alone. We observed dynamic changes in glycosaminoglycans at Day 1 and 7 post-TBI, with heparan sulfate, chondroitin sulfate, and hyaluronan remaining significantly increased after a week vis-à-vis craniotomy-alone tissues. We did not observe appreciable changes in circulating glycosaminoglycans in mice after experimental TBI compared to craniotomy-alone nor in patients with TBI and severe polytrauma compared to control patients with mild injuries, suggesting increases in injury site glycosaminoglycans are driven by local synthesis. We subsequently performed an unbiased whole genome transcriptomics analysis on mouse brain tissues 7 days post-TBI and discovered a significant induction of hyaluronan synthase 2, glypican-3, and decorin. The functional role of decorin after injury was further examined through multimodal behavioral testing comparing wild-type and Dcn-/- mice. We discovered that genetic ablation of Dcn led to an overall negative effect of TBI on function, exacerbating motor impairments after TBI. Collectively, our results provide a spatiotemporal characterization of post-TBI glycosaminoglycan alterations in the brain ECM and support an important adaptive role for decorin upregulation after TBI.

Predictors of adverse outcomes in aged patients critically ill with COVID-19: a retrospective study.

Older patients represent an inordinate proportion of intensive care unit (ICU) admissions and ICU mortality associated with coronavirus disease 2019 (COVID-19). In this retrospective cohort study, we examine 198 patients, aged 18 years or older, admitted to the ICU from March to June 2020. We aim to understand the relationships between age, number of comorbidities, and independent living prior to admission on outcomes of mortality, length of stay, renal failure, respiratory failure, and shock. In this cohort, we find that overall mortality was associated with respiratory failure severity (for every decrease of P:F by 50, odds ratio (OR) 2.98 (1.65-6.08)), acute renal failure (OR 4.61 (1.2-19.7)), and age 65 or greater (OR: 3.7 (1.86-7.36)). Surprisingly, increasing age was associated with less severe respiratory failure (R = 0.22, p < 0.01). When adjusting for pre-existing chronic kidney disease, age was not associated with development of acute kidney injury (OR: 1.01 (0.99-1.03)). While chronologic age is associated with mortality, it is not associated independently with severe end organ damage. This is consistent with growing evidence suggesting that a complex interplay between multimorbidity, immunosenescence, and physiologic age is primarily responsible for the vulnerability to COVID-19.

Aging-associated augmentation of gut microbiome virulence capability drives sepsis severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host, but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition, but an overabundance of genomic virulence factors that have functional consequence on host immune evasion. One Sentence Summary: The severity of sepsis in the aged host is in part mediated by longevity-associated increases in gut microbial virulence.

Aging-Associated Augmentation of Gut Microbiome Virulence Capability Drives Sepsis Severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition but also an overabundance of genomic virulence factors that have functional consequence on host immune evasion. IMPORTANCE Older adults suffer more frequent and worse outcomes from sepsis, a critical illness secondary to infection. The reasons underlying this unique susceptibility are incompletely understood. Prior work in this area has focused on how the immune response changes with age. The current study, however, focuses instead on alterations in the community of bacteria that humans live with within their gut (i.e., the gut microbiome). The central concept of this paper is that the bacteria in our gut evolve along with the host and "age," making them more efficient at causing sepsis.

Endothelial glycocalyx degradation during sepsis: Causes and consequences.

The glycocalyx is a ubiquitous structure found on endothelial cells that extends into the vascular lumen. It is enriched in proteoglycans, which are proteins attached to the glycosaminoglycans heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. In health and disease, the endothelial glycocalyx is a central regulator of vascular permeability, inflammation, coagulation, and circulatory tonicity. During sepsis, a life-threatening syndrome seen commonly in hospitalized patients, the endothelial glycocalyx is degraded, significantly contributing to its many clinical manifestations. In this review we discuss the intrinsically linked mechanisms responsible for septic endothelial glycocalyx destruction: glycosaminoglycan degradation and proteoglycan cleavage. We then examine the consequences of local endothelial glycocalyx loss to several organ systems and the systemic consequences of shed glycocalyx constituents. Last, we explore clinically relevant non-modifiable and modifiable factors that exacerbate or protect against endothelial glycocalyx shedding during sepsis.

Circulating heparan sulfate fragments mediate septic cognitive dysfunction.

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.

Intravital Microscopy in the Mouse Lung.

While reductionist in vitro approaches have allowed for careful interrogation of cellular pathways that underlie innate immune responses, they often fail to capture the complex multicellular interactions characteristic of acute inflammation. Intravital microscopy, by directly observing alveolar cell-cell interactions, provides unique insight into the complex intercellular mechanisms responsible for alveolar inflammation. This review discusses multiple potential approaches to intravital pulmonary imaging, with specific attention to in vivo microscopy of the freely moving mouse lung.