Katherine H. Overdier

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

The Circulating Glycosaminoglycan Signature of Respiratory Failure in Critically Ill Adults

Background: Endothelial glycocalyx degradation contributes to the pathogenesis of critical illness. Results: Mechanically ventilated subjects exhibited plasma glycocalyx breakdown signatures (glycosaminoglycan fragments) characteristic of direct versus indirect etiologies of respiratory failure. Conclusion: Circulating glycosaminoglycans provide insight into respiratory failure pathophysiology. Significance: This is the first study to characterize circulating glycosaminoglycans during critical illness, offering insight into the mechanisms underlying respiratory failure. Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated “human only” (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6 months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Rapid Automated Microscopy for Microbiological Surveillance of Ventilator-associated Pneumonia

RATIONALE Diagnosis of ventilator-associated pneumonia (VAP) is imprecise. OBJECTIVES To (1) determine whether alternate-day surveillance mini-bronchoalveolar lavage (mini-BAL) in ventilated adults could reduce time to initiation of targeted treatment and (2) evaluate the potential for automated microscopy to reduce analysis time. METHODS Adult intensive care unit patients who were anticipated to require ventilation for at least a further 48 hours were included. Mini-BALs were processed for identification, quantitation, and antibiotic susceptibility, using (1) clinical culture (50 ± 7 h) and (2) automated microscopy (∼5 h plus offline analysis). MEASUREMENTS AND MAIN RESULTS Seventy-seven mini-BALs were performed in 33 patients. One patient (3%) was clinically diagnosed with VAP. Of 73 paired samples, culture identified 7 containing pneumonia panel bacteria (>10(4) colony-forming units/ml) from five patients (15%) (4 Staphylococcus aureus [3 methicillin-resistant S. aureus], 2 Stenotrophomonas maltophilia, 1 Klebsiella pneumoniae) and resulted in antimicrobial changes/additions to two of five (40%) of those patients. Microscopy identified 7 of 7 microbiologically positive organisms and 64 of 66 negative samples compared with culture. Antimicrobial responses were concordant in four of five comparisons. Antimicrobial changes/additions would have occurred in three of seven microscopy-positive patients (43%) had those results been clinically available in 5 hours, including one patient diagnosed later with VAP despite negative mini-BAL cultures. CONCLUSIONS Microbiological surveillance detected infection in patients at risk for VAP independent of clinical signs, resulting in changes to antimicrobial therapy. Automated microscopy was 100% sensitive and 97% specific for high-risk pneumonia organisms compared with clinical culturing. Rapid microscopy-based surveillance may be informative for treatment and antimicrobial stewardship in patients at risk for VAP.

Analysis of Total Human Urinary Glycosaminoglycan Disaccharides by Liquid Chromatography–Tandem Mass Spectrometry

The determination of complex analytes, present at low concentrations, in biological fluids poses a difficult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography-tandem mass spectrometry to rapidly determine low concentrations of glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of glycosaminoglycan disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can subtly alter glycosaminoglycan content and composition.

Urinary Glycosaminoglycans Predict Outcomes in Septic Shock and Acute Respiratory Distress Syndrome

RATIONALE Degradation of the endothelial glycocalyx, a glycosaminoglycan (GAG)-rich layer lining the vascular lumen, is associated with the onset of kidney injury in animal models of critical illness. It is unclear if similar pathogenic degradation occurs in critically ill patients. OBJECTIVES To determine if urinary indices of GAG fragmentation are associated with outcomes in patients with critical illnesses such as septic shock or acute respiratory distress syndrome (ARDS). METHODS We prospectively collected urine from 30 patients within 24 hours of admission to the Denver Health Medical Intensive Care Unit (ICU) for septic shock. As a nonseptic ICU control, we collected urine from 25 surgical ICU patients admitted for trauma. As a medical ICU validation cohort, we obtained serially collected urine samples from 70 patients with ARDS. We performed mass spectrometry on urine samples to determine GAG (heparan sulfate, chondroitin sulfate, and hyaluronic acid) concentrations as well as patterns of heparan sulfate/chondroitin sulfate disaccharide sulfation. We compared these indices to measurements obtained using dimethylmethylene blue, an inexpensive, colorimetric urinary assay of sulfated GAGs. MEASUREMENTS AND MAIN RESULTS In septic shock, indices of GAG fragmentation correlated with both the development of renal dysfunction over the 72 hours after urine collection and with hospital mortality. This association remained after controlling for severity of illness and was similarly observed using the inexpensive dimethylmethylene blue assay. These predictive findings were corroborated using urine samples previously collected at three consecutive time points from patients with ARDS. CONCLUSIONS Early indices of urinary GAG fragmentation predict acute kidney injury and in-hospital mortality in patients with septic shock or ARDS. Clinical trial registered with www.clinicaltrials.gov (NCT01900275).

Innate Immune Function and Organ Failure Recovery in Adults With Sepsis

Purpose: Sepsis stimulates pro- and anti-inflammatory immune responses. The innate immune response is critical to organ injury repair. We tested for an association between innate immune function and organ function recovery in a prospective cohort of immune-competent adults with sepsis. Methods: We conducted a prospective observational cohort study enrolling immune-competent adults with sepsis. We tested innate immune function by quantification of lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production capacity in whole blood samples on hospital days 1, 4, and 6. The primary outcome was organ function recovery on day 4 defined as a 4-point decrease in the composite cardiovascular and respiratory Sequential Organ Failure Assessment (SOFA) score components or a SOFA score ≤2. Results: Patients with sepsis who recovered organ function by day 4 (n = 11) had similar baseline characteristics when compared to those with ongoing organ failure (n = 13). Tumor necrosis factor α production capacity was similar between the 2 groups on hospital days 1 and 4 but significantly different on day 6. Patients who regained organ function recovery had significantly higher TNF-α production capacity on day 6 (P = .01), which persisted after adjustment for age, Acute Physiology and Chronic Health Evaluation III score, and steroid administration (P = .03). There was no difference in TNF-α production capacity over time in those who survived to hospital discharge versus nonsurvivors. Conclusion: Increasing TNF-α production capacity is associated with improved organ failure recovery. Further studies are needed to evaluate a causal association between innate immune suppression and organ failure recovery as well as predictive accuracy for hospital survival. Impaired TNF-α production as a marker of sepsis-associated innate immune dysfunction may be a feasible target for immune stimulation to decrease time to organ failure recovery.