Lipidomic changes in a novel sepsis outcome-based analysis reveals potent pro-inflammatory and pro-resolving signaling lipids.

The purpose of this study was to investigate changes in the lipidome of patients with sepsis to identify signaling lipids associated with poor outcomes that could be linked to future therapies. Adult patients with sepsis were enrolled within 24h of sepsis recognition. Patients meeting Sepsis-3 criteria were enrolled from the emergency department or intensive care unit and blood samples were obtained. Clinical data were collected and outcomes of rapid recovery, chronic critical illness (CCI), or early death were adjudicated by clinicians. Lipidomic analysis was performed on two platforms, the Sciex™ 5500 device to perform a lipidomic screen of 1450 lipid species and a targeted signaling lipid panel using liquid-chromatography tandem mass spectrometry. For the lipidomic screen, there were 274 patients with sepsis: 192 with rapid recovery, 47 with CCI, and 35 with early deaths. CCI and early death patients were grouped together for analysis. Fatty acid (FA) 12:0 was decreased in CCI/early death, whereas FA 17:0 and 20:1 were elevated in CCI/early death, compared to rapid recovery patients. For the signaling lipid panel analysis, there were 262 patients with sepsis: 189 with rapid recovery, 45 with CCI, and 28 with early death. Pro-inflammatory signaling lipids from ω-6 poly-unsaturated fatty acids (PUFAs), including 15-hydroxyeicosatetraenoic (HETE), 12-HETE, and 11-HETE (oxidation products of arachidonic acid [AA]) were elevated in CCI/early death patients compared to rapid recovery. The pro-resolving lipid mediator from ω-3 PUFAs, 14(S)-hydroxy docosahexaenoic acid (14S-HDHA), was also elevated in CCI/early death compared to rapid recovery. Signaling lipids of the AA pathway were elevated in poor-outcome patients with sepsis and may serve as targets for future therapies.

Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair.

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-ß1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

The Lipid Intensive Drug Therapy for Sepsis Phase II Pilot Clinical Trial.

OBJECTIVES: Low cholesterol levels in early sepsis patients are associated with mortality. We sought to test if IV lipid emulsion administration to sepsis patients with low cholesterol levels would prevent a decline or increase total cholesterol levels at 48 hours. DESIGN: Phase II, adaptive, randomized pilot clinical trial powered for 48 patients. SETTING: Emergency department or ICU of an academic medical center. PATIENTS: Sepsis patients (first 24 hr) with Sequential Organ Failure Assessment greater than or equal to 4 or shock. INTERVENTIONS: Patients meeting study criteria, including screening total cholesterol levels less than or equal to 100 mg/dL or high-density lipoprotein cholesterol (HDL-C) + low-density lipoprotein cholesterol (LDL-C) less than or equal to 70 mg/dL, were randomized to receive one of three doses of lipid emulsion administered twice in 48 hours or no drug (controls). The primary endpoint was a change in serum total cholesterol (48 hr - enrollment) between groups. MEASUREMENTS AND MAIN RESULTS: Forty-nine patients were enrolled and randomized. Two patients randomized to lipid emulsion were withdrawn before drug administration. Data for 24 control patients and 23 lipid emulsion patients were analyzed. The mean change in total cholesterol from enrollment to 48 hours was not different between groups and was 5 mg/dL (sd 20) for lipid emulsion patients, and 2 mg/dL (sd 18) for control patients (p = 0.62). The mean changes in HDL-C and LDL-C were similar between groups. Mean change in triglycerides was elevated in lipid emulsion patients (61 mg/dL, sd 87) compared with controls (20 mg/dL, sd 70, p = 0.086). The 48-hour change in SOFA score was -2 (interquartile range [IQR] -4, -1) for control patients and -2 (IQR -3, 0) for lipid emulsion patients (p = 0.46). CONCLUSIONS: Administration of IV lipid emulsion to early sepsis patients with low cholesterol levels did not influence change in cholesterol levels from enrollment to 48 hours.

Early Physician Gestalt Versus Usual Screening Tools for the Prediction of Sepsis in Critically Ill Emergency Patients.

STUDY OBJECTIVE: Compare physician gestalt to existing screening tools for identifying sepsis in the initial minutes of presentation when time-sensitive treatments must be initiated. METHODS: This prospective observational study conducted with consecutive encounter sampling took place in the emergency department (ED) of an academic, urban, safety net hospital between September 2020 and May 2022. The study population included ED patients who were critically ill, excluding traumas, transfers, and self-evident diagnoses. Emergency physician gestalt was measured using a visual analog scale (VAS) from 0 to 100 at 15 and 60 minutes after patient arrival. The primary outcome was an explicit sepsis hospital discharge diagnosis. Clinical data were recorded for up to 3 hours to compare Systemic Inflammatory Response Syndrome (SIRS), Sequential Organ Failure Assessment (SOFA), quick SOFA (qSOFA), Modified Early Warning Score (MEWS), and a logistic regression machine learning model using Least Absolute Shrinkage and Selection Operator (LASSO) for variable selection. The screening tools were compared using receiver operating characteristic analysis and area under the curve calculation (AUC). RESULTS: A total of 2,484 patient-physician encounters involving 59 attending physicians were analyzed. Two hundred seventy-five patients (11%) received an explicit sepsis discharge diagnosis. When limited to available data at 15 minutes, initial VAS (AUC 0.90; 95% confidence interval [CI] 0.88, 0.92) outperformed all tools including LASSO (0.84; 95% CI 0.82 to 0.87), qSOFA (0.67; 95% CI 0.64 to 0.71), SIRS (0.67; 95% 0.64 to 0.70), SOFA (0.67; 95% CI 0.63 to 0.70), and MEWS (0.66; 95% CI 0.64 to 0.69). Expanding to data available at 60 minutes did not meaningfully change results. CONCLUSION: Among adults presenting to an ED with an undifferentiated critical illness, physician gestalt in the first 15 minutes of the encounter outperformed other screening methods in identifying sepsis.

TRENDS IN CHOLESTEROL AND LIPOPROTEINS ARE ASSOCIATED WITH ACUTE RESPIRATORY DISTRESS SYNDROME INCIDENCE AND DEATH AMONG SEPSIS PATIENTS.

ABSTRACT: Objective: Compare changes in cholesterol and lipoprotein levels occurring in septic patients with and without acute respiratory distress syndrome (ARDS) and by survivorship. Methods: We reanalyzed data from prospective sepsis studies. Cholesterol and lipoprotein levels were analyzed using univariate testing to detect changes between septic patients with or without ARDS, and among ARDS survivors compared with nonsurvivors at enrollment (first 24 h of sepsis) and 48 to 72 h later. Results: 214 patients with sepsis were included of whom 48 had ARDS and 166 did not have ARDS. Cholesterol and lipoproteins among septic ARDS versus non-ARDS showed similar enrollment levels. However, 48 to 72 h after enrollment, change in median total cholesterol (48/72 h - enrollment) was significantly different between septic ARDS (-4, interquartile range [IQR] -23.5, 6.5, n = 35) and non-ARDS (0, -10.0, 17.5, P = 0.04; n = 106). When compared by ARDS survivorship, ARDS nonsurvivors (n = 14) had lower median total cholesterol levels (75.5, IQR 68.4, 93.5) compared with ARDS survivors (113.0, IQR 84.0, 126.8, P = 0.022), and lower median enrollment low-density lipoprotein cholesterol (LDL-C) levels (27, IQR 19.5-34.5) compared with ARDS survivors (43, IQR 27-67, P = 0.013; n = 33). Apolipoprotein A-I levels were also significantly lower in ARDS nonsurvivors (n = 14) (87.6, IQR 76.45-103.64) compared with ARDS survivors (130.0, IQR 73.25-165.47, P = 0.047; n = 33). At 48 to 72 h, for ARDS nonsurvivors, median levels of low-density lipoprotein cholesterol (9.0, IQR 4.3, 18.0; n = 10), LDL-C (17.0, IQR 5.0, 29.0; n = 9), and total cholesterol (59.0, 45.3, 81.5; n = 10) were significantly lower compared with ARDS survivors' (n = 25) levels of low-density lipoprotein cholesterol (20.0, IQR 12.0-39.0, P = 0.014), LDL-C (42.0, IQR 27.0-58.0, P = 0.019), and total cholesterol (105.0, IQR 91.0, 115.0, P = 0.003). Conclusions: Change in total cholesterol was different in septic ARDS versus non-ARDS. Total cholesterol, LDL-C, and apolipoprotein A-I levels were lower in ARDS nonsurvivors compared with survivors. Future studies of dysregulated cholesterol metabolism in septic ARDS patients are needed to understand biology and links to potential therapies.

Racial disparities in septic shock mortality: a retrospective cohort study.

Background: Patients with septic shock have the highest risk of death from sepsis, however, racial disparities in mortality outcomes in this cohort have not been rigorously investigated. Our objective was to describe the association between race/ethnicity and mortality in patients with septic shock. Methods: Our study is a retrospective cohort study of adult patients in the OneFlorida Data Trust (Florida, United States of America) admitted with septic shock between January 2012 and July 2018. We identified patients as having septic shock if they received vasopressors during their hospital encounter and had either an explicit International Classification of Disease (ICD) code for sepsis, or had an infection ICD code and received intravenous antibiotics. Our primary outcome was 90-day mortality. Our secondary outcome was in-hospital mortality. Multiple logistic regression with Least Absolute Shrinkage and Selection Operator (LASSO) for variable selection was used to assess associations. Findings: There were 13,932 patients with septic shock in our cohort. The mean age was 61 years (SD 16), 68% of the cohort identified as White (n = 9419), 28% identified as Black (n = 3936), 2% (n = 294) identified as Hispanic ethnicity, and 2% as other races not specified in the previous groups (n = 283). In our logistic regression model for 90-day mortality, patients identified as Black had 1.57 times the odds of mortality (95% CI 1.07-2.29, p = 0.02) compared to White patients. Other significant predictors included mechanical ventilation (OR 3.66, 95% CI 3.35-4.00, p < 0.01), liver disease (OR 1.75, 95% CI 1.59-1.93, p < 0.01), laboratory components of the Sequential Organ Failure Assessment score (OR 1.18, 95% CI 1.16-1.21, p < 0.01), lactate (OR 1.10, 95% CI 1.08-1.12, p < 0.01), congestive heart failure (OR 1.19, 95% CI 1.10-1.30, p < 0.01), human immunodeficiency virus (OR 1.35, 95% CI 1.04-1.75, p = 0.03), age (OR 1.04, 95% CI 1.04-1.04, p < 0.01), and the interaction between age and race (OR 0.99, 95% CI 0.99-1.00, p < 0.01). Among younger patients (<45 years), patients identified as Black accounted for a higher proportion of the deaths. Results were similar in the in-hospital mortality model. Interpretation: In this retrospective study of septic shock patients, we found that patients identified as Black had higher odds of mortality compared to patients identified as non-Hispanic White. Our findings suggest that the greatest disparities in mortality are among younger Black patients with septic shock. Funding: National Institutes of Health National Center for Advancing Translational Sciences (1KL2TR001429); National Institute of Health National Institute of General Medical Sciences (1K23GM144802).

Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

DHCR7 Expression Predicts Poor Outcomes and Mortality From Sepsis.

This is a study of lipid metabolic gene expression patterns to discover precision medicine for sepsis. OBJECTIVES: Sepsis patients experience poor outcomes including chronic critical illness (CCI) or early death (within 14 d). We investigated lipid metabolic gene expression differences by outcome to discover therapeutic targets. DESIGN SETTING AND PARTICITPANTS: Secondary analysis of samples from prospectively enrolled sepsis patients (first 24 hr) and a zebrafish endotoxemia model for drug discovery. Patients were enrolled from the emergency department or ICU at an urban teaching hospital. Enrollment samples from sepsis patients were analyzed. Clinical data and cholesterol levels were recorded. Leukocytes were processed for RNA sequencing and reverse transcriptase polymerase chain reaction. A lipopolysaccharide zebrafish endotoxemia model was used for confirmation of human transcriptomic findings and drug discovery. MAIN OUTCOMES AND MEASURES: The derivation cohort included 96 patients and controls (12 early death, 13 CCI, 51 rapid recovery, and 20 controls) and the validation cohort had 52 patients (6 early death, 8 CCI, and 38 rapid recovery). RESULTS: The cholesterol metabolism gene 7-dehydrocholesterol reductase (DHCR7) was significantly up-regulated in both derivation and validation cohorts in poor outcome sepsis compared with rapid recovery patients and in 90-day nonsurvivors (validation only) and validated using RT-qPCR analysis. Our zebrafish sepsis model showed up-regulation of dhcr7 and several of the same lipid genes up-regulated in poor outcome human sepsis (dhcr24, sqlea, cyp51, msmo1, and ldlra) compared with controls. We then tested six lipid-based drugs in the zebrafish endotoxemia model. Of these, only the Dhcr7 inhibitor AY9944 completely rescued zebrafish from lipopolysaccharide death in a model with 100% lethality. CONCLUSIONS: DHCR7, an important cholesterol metabolism gene, was up-regulated in poor outcome sepsis patients warranting external validation. This pathway may serve as a potential therapeutic target to improve sepsis outcomes.

Association of Early Serum Phosphate Levels and Mortality in Patients with Sepsis.

BACKGROUND: Metabolic derangements in sepsis influence phosphate levels, which may predict mortality outcomes. We investigated the association between initial phosphate levels and 28-day mortality in patients with sepsis. METHODS: We conducted a retrospective analysis of patients with sepsis. Initial (first 24 hours) phosphate levels were divided into phosphate quartile groups for comparisons. We used repeated-measures mixed-models to assess differences in 28-day mortality across the phosphate groups, adjusting for other predictors identified by the Least Absolute Shrinkage and Selection Operator variable selection technique. RESULTS: A total of 1,855 patients were included with 13% overall 28-day mortality (n=237). The highest phosphate quartile (>4.0 milligrams per deciliter [mg/dL]) had a higher mortality rate (28%) than the three lower quartiles (P<0.001). After adjustment (age, organ failure, vasopressor administration, liver disease), the highest initial phosphate was associated with increased odds of 28-day mortality. Patients in the highest phosphate quartile had 2.4 times higher odds of death than the lowest (≤2.6 mg/dL) quartile (P<0.01), 2.6 times higher than the second (2.6-3.2 mg/dL) quartile (P<0.01), and 2.0 times higher than the third (3.2-4.0 mg/dL) quartile (P=0.04). CONCLUSION: Septic patients with the highest phosphate levels had increased odds of mortality. Hyperphosphatemia may be an early indicator of disease severity and risk of adverse outcomes from sepsis.

An ASO therapy for Angelman syndrome that targets an evolutionarily conserved region at the start of the UBE3A-AS transcript.

Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A (UBE3A) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense (UBE3A-AS) transcript, which represents the distal end of the small nucleolar host gene 14 (SNHG14) transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we described the development of an antisense oligonucleotide (ASO) therapy for Angelman syndrome that targets an evolutionarily conserved region demarcating the start of the UBE3A-AS transcript. We designed and chemically optimized gapmer ASOs targeting specific sequences at the start of the human UBE3A-AS transcript. We showed that ASOs targeting this region precisely and efficiently repress the transcription of UBE3A-AS, reactivating the expression of the paternal UBE3A allele in neurotypical and Angelman syndrome induced pluripotent stem cell-derived neurons. We further showed that human-targeted ASOs administered to the CNS of cynomolgus macaques by lumbar intrathecal injection repress UBE3A-AS and reactivate the expression of the paternal UBE3A allele throughout the CNS. These findings support the advancement of this investigational molecular therapy for Angelman syndrome into clinical development (ClinicalTrials.gov, NCT04259281).

DHCR7 Expression Predicts Poor Outcomes and Mortality from Sepsis.

Objective: Sepsis patients experience poor outcomes including chronic critical illness (CCI) or early death (within 14 days). We investigated lipid metabolic gene expression differences by outcome to discover therapeutic targets. Design: Secondary analysis of samples from prospectively enrolled sepsis patients and a zebrafish sepsis model for drug discovery. Setting: Emergency department or ICU at an urban teaching hospital. Patients: Sepsis patients presenting within 24 hours. Methods: Enrollment samples from sepsis patients were analyzed. Clinical data and cholesterol levels were recorded. Leukocytes were processed for RNA sequencing (RNA-seq) and reverse transcriptase polymerase chain reaction (RT-qPCR). A lipopolysaccharide (LPS) zebrafish sepsis model was used for confirmation of human transcriptomic findings and drug discovery. Measurements and Main Results: There were 96 samples in the derivation (76 sepsis, 20 controls) and 52 in the validation cohort (sepsis only). The cholesterol metabolism gene 7-Dehydrocholesterol Reductase ( DHCR7) was significantly upregulated in both derivation and validation cohorts in poor outcome sepsis compared to rapid recovery patients and in 90-day non-survivors (validation only) and validated using RT-qPCR analysis. Our zebrafish sepsis model showed upregulation of dhcr7 and several of the same lipid genes upregulated in poor outcome human sepsis (dhcr24, sqlea, cyp51, msmo1 , ldlra) compared to controls. We then tested six lipid-based drugs in the zebrafish sepsis model. Of these, only the Dhcr7 inhibitor AY9944 completely rescued zebrafish from LPS death in a model with 100% lethality. Conclusions: DHCR7, an important cholesterol metabolism gene, was upregulated in poor outcome sepsis patients warranting external validation. This pathway may serve as a potential therapeutic target to improve sepsis outcomes.

Association Between Hypocholesterolemia and Mortality in Critically Ill Patients With Sepsis: A Systematic Review and Meta-Analysis.

To ascertain the association between cholesterol and triglyceride levels on ICU admission and mortality in patients with sepsis. DATA SOURCES: Systematic review and meta-analysis of published studies on PubMed and Embase. STUDY SELECTION: All observational studies reporting ICU admission cholesterol and triglyceride levels in critically ill patients with sepsis were included. Authors were contacted for further data. DATA EXTRACTION: Eighteen observational studies were identified, including 1,283 patients with a crude overall mortality of 33.3%. Data were assessed using Revman (Version 5.1, Cochrane Collaboration, Oxford, United Kingdom) and presented as mean difference (MD) with 95% CIs, p values, and I 2 values. DATA SYNTHESIS: Admission levels of total cholesterol (17 studies, 1,204 patients; MD = 0.52 mmol/L [0.27-0.77 mmol/L]; p < 0.001; I 2 = 91%), high-density lipoprotein (HDL)-cholesterol (14 studies, 991 patients; MD = 0.08 mmol/L [0.01-0.15 mmol/L]; p = 0.02; I 2 = 61%), and low-density lipoprotein (LDL)-cholesterol (15 studies, 1,017 patients; MD = 0.18 mmol/L [0.04-0.32 mmol/L]; p = 0.01; I 2 = 71%) were significantly lower in eventual nonsurvivors compared with survivors. No association was seen between admission triglyceride levels and mortality (15 studies, 1,070 patients; MD = 0.00 mmol/L [-0.16 to 0.15 mmol/L]; p = -0.95; I 2 = 79%). CONCLUSIONS: Mortality was associated with lower levels of total cholesterol, HDL-cholesterol, and LDL-cholesterol, but not triglyceride levels, in patients admitted to ICU with sepsis. The impact of cholesterol replacement on patient outcomes in sepsis, particularly in at-risk groups, merits investigation.

RNA-based drug discovery for spinal muscular atrophy: a story of small molecules and antisense oligonucleotides.

INTRODUCTION: Spinal Muscular Atrophy (SMA), the second most prevalent autosomal genetic disease affecting infants, is caused by the lack of SMN1, which encodes a neuron functioning vital protein, SMN. Improving exon 7 splicing in the paralogous gene SMN2, also coding for SMN protein, increases protein production efficiency from SMN2 to overcome the genetic deficit in SMN1. Several molecular mechanisms have been investigated to improve SMN2 functional splicing. AREAS COVERED: This manuscript will cover two of the three mechanistically distinct available treatment options for SMA, both targeting the SMN2 splicing mechanism. The first therapeutic, nusinersen (Spinraza®, 2017), is an antisense oligonucleotide (ASO) targeting the splicing inhibitory sequence in the intron downstream of exon 7 from SMN2, thus increasing exon 7 inclusion. The second drug is a small molecule, risdiplam (Evrysdi®, 2021), that enhances the binding of splice factors and also promotes exon 7 inclusion. Both therapies, albeit through different mechanisms, increase full-length SMN protein expression. EXPERT OPINION: Nusinersen and risdiplam have directly helped SMA patients and families, but they also herald a sea change in drug development for genetic diseases. This piece aims to draw parallels between both development histories; this may help chart the course for future targeted agents.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Introduction: Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Method: Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. Results and discussion: CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Evaluation of rapid transepithelial electrical resistance (TEER) measurement as a metric of kidney toxicity in a high-throughput microfluidic culture system.

Rapid non-invasive kidney-specific readouts are essential to maximizing the potential of microfluidic tissue culture platforms for drug-induced nephrotoxicity screening. Transepithelial electrical resistance (TEER) is a well-established technique, but it has yet to be evaluated as a metric of toxicity in a kidney proximal tubule (PT) model that recapitulates the high permeability of the native tissue and is also suitable for high-throughput screening. We utilized the PREDICT96 high-throughput microfluidic platform, which has rapid TEER measurement capability and multi-flow control, to evaluate the utility of TEER sensing for detecting cisplatin-induced toxicity in a human primary PT model under both mono- and co-culture conditions as well as two levels of fluid shear stress (FSS). Changes in TEER of PT-microvascular co-cultures followed a dose-dependent trend similar to that demonstrated by lactate dehydrogenase (LDH) cytotoxicity assays and were well-correlated with tight junction coverage after cisplatin exposure. Additionally, cisplatin-induced changes in TEER were detectable prior to increases in cell death in co-cultures. PT mono-cultures had a less differentiated phenotype and were not conducive to toxicity monitoring with TEER. The results of this study demonstrate that TEER has potential as a rapid, early, and label-free indicator of toxicity in microfluidic PT-microvascular co-culture models.

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models.

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

Right Ventricular Outflow Tract Surgical Resection in Young, Large Animal Model for the Study of Alternative Cardiovascular Patches.

Tetralogy of Fallot (ToF) is a severe congenital heart defect (CHD) that requires surgical reconstruction soon after birth. Reconstructive surgery involves the implantation of synthetic cardiovascular patches to widen the right ventricular outflow tract (RVOT) and repair defects in the septal wall. However, synthetic patches can cause complications for these patients later in life as they do not integrate or adapt in the tissue of a growing patient; a limitation that could be solved with the development of a patch fabricated from a degradable biomaterial. Unfortunately, the lack of appropriate pre-clinical models has hindered the development of novel patch materials. Currently, most studies use rodent models to study the efficacy of new patch materials; however, large animal models are necessary to develop realistically sized patches in a clinically relevant growing heart where gradients in diffusion and length scales for cell migration are more similar to the human. Here, we describe a novel method by which a Satinsky vascular clamp is used to isolate RVOT muscle for resection followed by implantation of a cardiovascular patch in an appropriately young, rapidly growing porcine model.

Efficacy of Losartan in Hospitalized Patients With COVID-19-Induced Lung Injury: A Randomized Clinical Trial.

Importance: SARS-CoV-2 viral entry may disrupt angiotensin II (AII) homeostasis, contributing to COVID-19 induced lung injury. AII type 1 receptor blockade mitigates lung injury in preclinical models, although data in humans with COVID-19 remain mixed. Objective: To test the efficacy of losartan to reduce lung injury in hospitalized patients with COVID-19. Design, Setting, and Participants: This blinded, placebo-controlled randomized clinical trial was conducted in 13 hospitals in the United States from April 2020 to February 2021. Hospitalized patients with COVID-19 and a respiratory sequential organ failure assessment score of at least 1 and not already using a renin-angiotensin-aldosterone system (RAAS) inhibitor were eligible for participation. Data were analyzed from April 19 to August 24, 2021. Interventions: Losartan 50 mg orally twice daily vs equivalent placebo for 10 days or until hospital discharge. Main Outcomes and Measures: The primary outcome was the imputed arterial partial pressure of oxygen to fraction of inspired oxygen (Pao2:Fio2) ratio at 7 days. Secondary outcomes included ordinal COVID-19 severity; days without supplemental o2, ventilation, or vasopressors; and mortality. Losartan pharmacokinetics and RAAS components (AII, angiotensin-[1-7] and angiotensin-converting enzymes 1 and 2)] were measured in a subgroup of participants. Results: A total of 205 participants (mean [SD] age, 55.2 [15.7] years; 123 [60.0%] men) were randomized, with 101 participants assigned to losartan and 104 participants assigned to placebo. Compared with placebo, losartan did not significantly affect Pao2:Fio2 ratio at 7 days (difference, -24.8 [95%, -55.6 to 6.1]; P = .12). Compared with placebo, losartan did not improve any secondary clinical outcomes and led to fewer vasopressor-free days than placebo (median [IQR], 9.4 [9.1-9.8] vasopressor-free days vs 8.7 [8.2-9.3] vasopressor-free days). Conclusions and Relevance: This randomized clinical trial found that initiation of orally administered losartan to hospitalized patients with COVID-19 and acute lung injury did not improve Pao2:Fio2 ratio at 7 days. These data may have implications for ongoing clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04312009.

Self-Assembled Human Skin Equivalents Model Macrophage Activation of Cutaneous Fibrogenesis in Systemic Sclerosis.

OBJECTIVE: The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis. METHODS: SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor ß (TGFß) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFß blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE. CONCLUSION: These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFß activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.