Analysis of Cerebrospinal Fluid, Plasma ß-Amyloid Biomarkers, and Cognition from a 2-Year Phase 2 Trial Evaluating Oral ALZ-801/Valiltramiprosate in APOE4 Carriers with Early Alzheimer's Disease Using Quantitative Systems Pharmacology Model.

INTRODUCTION: ALZ-801/valiltramiprosate is an oral, small-molecule inhibitor of beta-amyloid (Aß) aggregation and oligomer formation in late-stage development as a disease-modifying therapy for early Alzheimer's disease (AD). The present investigation provides a quantitative systems pharmacology (QSP) analysis of amyloid fluid biomarkers and cognitive results from a 2-year ALZ-801 Phase 2 trial in APOE4 carriers with early AD. METHODS: The single-arm, open-label phase 2 study evaluated effects of ALZ-801 265 mg two times daily (BID) on cerebrospinal fluid (CSF) and plasma amyloid fluid biomarkers over 104 weeks in APOE4 carriers with early AD [Mini-Mental State Examination (MMSE) ≥ 22]. Subjects with positive CSF biomarkers for amyloid (Aß42/Aß40) and tau pathology (p-tau181) were enrolled, with serial CSF and plasma levels of Aß42 and Aß40 measured over 104 weeks. Longitudinal changes of CSF Aß42, plasma Aß42/Aß40 ratio, and cognitive Rey Auditory Verbal Learning Test (RAVLT) were compared with the established natural disease trajectories in AD using a QSP approach. The natural disease trajectory data for amyloid biomarkers and RAVLT were extracted from a QSP model and an Alzheimer's disease neuroimaging initiative population model, respectively. Analyses were stratified by disease severity and sex. RESULTS: A total of 84 subjects were enrolled. Excluding one subject who withdrew at the early stage of the trial, data from 83 subjects were used for this analysis. The ALZ-801 treatment arrested the progressive decline in CSF Aß42 level and plasma Aß42/Aß40 ratio, and stabilized RAVLT over 104 weeks. Both sexes showed comparable responses to ALZ-801, whereas mild cognitive impairment (MCI) subjects (MMSE ≥ 27) exhibited a larger biomarker response compared with more advanced mild AD subjects (MMSE 22-26). CONCLUSIONS: In this genetically defined and biomarker-enriched early AD population, the QSP analysis demonstrated a positive therapeutic effect of oral ALZ-801 265 mg BID by arresting the natural decline of monomeric CSF and plasma amyloid biomarkers, consistent with the target engagement to prevent their aggregation into soluble neurotoxic oligomers and subsequently into insoluble fibrils and plaques over 104 weeks. Accompanying the amyloid biomarker changes, ALZ-801 also stabilized the natural trajectory decline of the RAVLT memory test, suggesting that the clinical benefits are consistent with its mechanism of action. This sequential effect arresting the disease progression on biomarkers and cognitive decline was more pronounced in the earlier symptomatic stages of AD. The QSP analysis provides fluid biomarker and clinical evidence for ALZ-801 as a first-in-class, oral small-molecule anti-Aß oligomer agent with disease modification potential in AD. TRIAL REGISTRY: https://clinicaltrials.gov/study/NCT04693520.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 µmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

The Risk of Hemorrhagic Complications After Anticoagulation Therapy in Trauma Patients: A Multicenter Evaluation.

BACKGROUND: The use of anticoagulation therapy (ACT) in trauma patients during the post-injury period presents a challenge given the increased risk of hemorrhage. Guidelines regarding whether and when to initiate ACT are lacking, and as a result, practice patterns vary widely. The purpose of this study is to describe the incidence of hemorrhagic complications in patients who received ACT during their hospitalization, identify risk factors, and characterize the required interventions. METHODS: In this retrospective cohort study, all trauma admissions at two Level 1 trauma centers between January 2015 and December 2020 were reviewed. Patients with pre-existing ACT use or those who developed a new indication for ACT were included for analysis. Demographic and outcome data were collected for those who received ACT during their admission. Comparisons were then made between the complications and no complications groups. A subgroup analysis was performed for all patients started on ACT within 14 days of injury. RESULTS: A total of 812 patients were identified as having an indication for ACT, and 442 patients received ACT during the post-injury period. The overall incidence of hemorrhagic complications was 12.7%. Of those who sustained hemorrhagic complications, 18 required procedural intervention. On regression analysis, male sex, severe injuries, and the need for hemorrhage control surgery on arrival were all found to be associated with hemorrhagic complications after the initiation of ACT. Waiting 7-14 days from the time of injury to initiate ACT reduced the odds of complications by 46% and 71%, respectively. CONCLUSIONS: The use of ACT in trauma during the post-injury period is not without risk. Waiting 7-14 days post-injury might greatly reduce the risk of hemorrhagic complications. STUDY TYPE/LEVEL OF EVIDENCE: Therapeutic/care management study: Level IV.

Plasma AGE and Oxidation Products, Renal Function, and Preeclampsia in Pregnant Women with Type 1 Diabetes: A Prospective Observational Study.

Aims: We aimed to determine whether plasma advanced glycation end products or oxidation products (AGE/oxidation-P) predict altered renal function and/or preeclampsia (PE) in pregnant women with type 1 diabetes. Methods: Prospectively, using a nested case-control design, we studied 47 pregnant women with type 1 diabetes, of whom 23 developed PE and 24 did not. Nineteen nondiabetic, normotensive pregnant women provided reference values. In plasma obtained at ~12, 22, and 32 weeks' gestation (visits 1, 2, and 3; V1-V3), we measured five AGE products (carboxymethyllysine (CML), carboxyethyl-lysine (CEL), methylglyoxal-hydroimidazolone (MGH1), 3-deoxyglucosone hydroimidazolone (3DGH), and glyoxal-hydroimidazolone (GH1)) and four oxidation products (methionine sulfoxide (MetSO), 2-aminoadipic acid (2-AAA), 3-nitrotyrosine (3NT), and dityrosine (DT)), by liquid chromatography/mass spectroscopy. Clinical outcomes were "estimated glomerular filtration rate" (eGFR) at each visit and onset of PE. Results: In diabetic women, associations between AGE/oxidation-P and eGFR were found only in those who developed PE. In this group, CEL, MGH1, and GH1 at V2 and CML, CEL, MGH1, and GH1 at V3 were inversely associated with contemporaneous eGFR, while CEL, MGH1, 3DGH, and GH1 at V2 were inversely associated with eGFR at V3 (all p < 0.05). There were no associations of plasma AGE or oxidation-P with pregnancy-related development of proteinuria or PE. Conclusions: Inverse associations of second and early third trimester plasma AGE with eGFR among type 1 diabetic women who developed PE suggest that these patients constitute a subset susceptible to AGE-mediated injury and thus to cardiorenal complications later in life. However, AGE/oxidation-P did not predict PE in type 1 diabetic women.

Resting heart rate (variability) and cognition relationships reveal cognitively healthy individuals with pathological amyloid/tau ratio.

Introduction: Resting heart rate (HR) and heart rate variability (HRV) have been linked with cognition in the general population and in older individuals. The knowledge of this aspect of heart-brain relationship is relatively absent in older individuals with early Alzheimer's disease (AD) pathology. This study explores relationships of the HR, HRV, and cognition in cognitively healthy individuals with pathological amyloid/tau ratio (CH-PATs) in cerebral spinal fluid (CSF) compared to those with normal ratio (CH-NATs). Methods: We examined therelationshipsbetween1) resting HR and Mini-Mental State Examination (MMSE); 2) resting HR and brain processing during Stroop interference; and 3) resting vagally mediated HRV (vmHRV) and task switching performance. Results: Our studies showed that compared to CH-NATs, those CH-PATs with higher resting HR presented with lower MMSE, and less brain activation during interference processing. In addition, resting vmHRV was significantly correlated with task switching accuracy in CH-NATs, but not in CH-PATs. Discussion: Thesethreedifferenttestsindicatedysfunctionalheart-brainconnections in CH-PATs, suggesting a potential cardio-cerebral dysfunctional integration.

Adjuvant chemotherapy, not radiotherapy, prolongs survival for node-negative non-small cell lung cancer with positive surgical margins.

Objective: The study objective was to determine differences in survival depending on adjuvant therapy type, timing, and sequence in node-negative disease with positive margins after non-small cell lung cancer resection. Methods: The National Cancer Database was queried for patients with positive margins after surgical resection of treatment-naïve cT1-4N0M0 pN0 non-small cell lung cancer who underwent adjuvant radiotherapy or chemotherapy from 2010 to 2016. Adjuvant treatment groups were defined as surgery alone, chemotherapy alone, radiotherapy alone, concurrent chemoradiotherapy, sequential chemotherapy then radiotherapy, and sequential radiotherapy then chemotherapy. The impact of adjuvant radiotherapy initiation timing on survival was evaluated using multivariable Cox regression. Kaplan-Meier curves were generated to compare 5-year survival. Results: A total of 1713 patients met inclusion criteria. Five-year survival estimates differed significantly between cohorts: surgery alone, 40.7%; chemotherapy alone, 47.0%; radiotherapy alone, 35.1%; concurrent chemoradiotherapy, 45.7%; sequential chemotherapy then radiotherapy, 36.6%; and sequential radiotherapy then chemotherapy, 32.2% (P = .033). Compared with surgery alone, adjuvant radiotherapy alone had a lower estimated survival at 5 years, although overall survival did not differ significantly (P = .8). Chemotherapy alone improved 5-year survival compared with surgery alone (P = .0016) and provided a statistically significant survival advantage over adjuvant radiotherapy (P = .002). Compared with radiotherapy-inclusive multimodal therapies, chemotherapy alone yielded similar 5-year survival (P = .066). Multivariable Cox regression showed an inverse linear association between time to adjuvant radiotherapy initiation and survival, but with an insignificant trend (10-day hazard ratio, 1.004; P = .90). Conclusions: In treatment-naïve cT1-4N0M0 pN0 non-small cell lung cancer with positive surgical margins, only adjuvant chemotherapy was associated with a survival improvement compared with surgery alone, with no radiotherapy-inclusive treatment providing additional survival benefit. Delayed timing of radiotherapy initiation was not associated with a survival reduction.

Identifying Populations at Risk for Difficulty Managing Emotions and Social Interactions After Burn Injury.

There has been conflicting data on the relationship between burn severity and psychological outcomes. The present study aims to characterize the baseline psychosocial disposition of adults attending outpatient burn clinic at a large urban safety net hospital, as well as the impact of clinical course on self-reported psychosocial well-being. Adult patients attending outpatient burn clinic completed survey questions from the National Institutes of Health Patient-Reported Outcomes Measurement Information System Managing Chronic Conditions: Self-Efficacy for Managing Social Interactions (SEMSI-4) and Managing Emotions (SEME). Sociodemographic variables were collected from surveys and retrospective chart review. Clinical variables included total body surface area burned, initial hospital length of stay, surgical history, and days since injury. Poverty level was estimated by U.S. census data using patient's home ZIP code. Scores on SEME-4 and SEMSI-4 were compared to the population mean by one-sample T-test, and independent variables evaluated for associations with managing emotions and social interactions by Tobit regression while adjusting for demographic variables. The 71 burn patients surveyed had lower scores in SEMSI-4 (mean = 48.0, P = .041) but not SEME-4 (mean = 50.9, P = .394) versus the general population. Marital status and neighborhood poverty level were associated with SEMSI-4, while length of stay and % total body surface area burned were associated with SEME-4. Patients who are single or from poorer neighborhoods may have difficulty interacting with their environment after burn injury and need extra social support. Prolonged hospitalization and increased severity of burn injury may have more impact on emotional regulation; these patients may benefit from psychotherapy during recovery.

Race and Ethnicity Influences Outcomes of Adult Burn Patients.

Outcomes of burn survivors is a growing field of interest; however, there is little data comparing the outcomes of burn survivors by ethnicity. This study seeks to identify any inequities in burn outcomes by racial and ethnic groups. A retrospective chart review of an ABA Certified burn center at a large urban safety net hospital identified adult inpatient admissions from 2015 to 2019. A total of 1142 patients were categorized by primary ethnicity: 142 black or African American, 72 Asian, 479 Hispanic or Latino, 90 white, 215 other, and 144 patients whose race or ethnicity was unrecorded. Multivariable analyses evaluated the relationship between race and ethnicity and outcomes. Covariate confounders were controlled by adjustment of demographic, social, and prehospital clinical factors to isolate differences that might not be explained by other factors. After controlling for covariates, black patients had 29% longer hospital stays (P = .043). Hispanic patients were more likely to be discharged to home or to hospice care (P = .005). Hispanic ethnicity was associated with a 44% decrease in the odds of discharge to acute care, inpatient rehabilitation, or a ward outside the burn unit (P = .022). Black and Hispanic patients had a higher relative chance of having publicly assisted insurance, versus private insurance, than their white counterparts (P = .041, P = .011 respectively). The causes of these inequities are indeterminate. They may stem from socioeconomic status not entirely accounted for, ethnic differences in comorbidity related to stressors, or inequity in health care delivery.

Heart transplantation for pediatric foreign nationals in the United States.

BACKGROUND: This study aimed to clarify survival outcomes, waitlist mortality, and waitlist days of heart transplantation of pediatric foreign nationals compared to pediatric United States (US) citizens. METHODS: We retrieved data from March 2012 to June 2021 in the United Network Organ Sharing (UNOS) registry. RESULTS: Of 5857 pediatric patients newly waitlisted, 133 (2.27%) patients were non-US citizen/non-US residents (non-citizen non-resident [NCNR]). Patients with congenital heart disease were higher in the US citizen group than in the NCNR group (51.9% vs. 22.6%, p < .001); 76.7% of patients in the NCNR group (102/133) had cardiomyopathy. Of the 133 NCNRs, 111 patients (83.5%) underwent heart transplantation, which was significantly higher than that in the US citizen group (68.6%, p < .001). The median waitlist time was 71 days (IQR, 22-172 days) in the NCNR group and 74 days (29-184 days) in the US citizen group (p = .48). Survival after heart transplant was significantly better in the NCNR group than in the US citizen group (n = 3982; logrank test p = .015). CONCLUSIONS: Heart transplantation for pediatric foreign nationals was mostly indicated for cardiomyopathy, and their transplant rate was significantly higher than that in the US citizen group, with better survival outcomes. The better survival outcomes in the NCNR group compared to the US citizen group can likely be attributed to the differing diagnoses for which transplantation was performed.

Peer Support Groups: Identifying Disparities to Improve Participation.

Disparities in psychosocial outcomes after burn injury exist in patients from racial or ethnic minority groups in the United States. Peer support groups can help patients with many psychosocial aspects of recovery from burns; however, access to such support among patients of racial and ethnic minority or low socioeconomic groups are unknown. The present study examined participation rates in outpatient peer support within this patient population. Patients attending outpatient clinic at an urban safety-net hospital and regional burn center with a majority minority patient population were asked about participation in burn survivor group, interest in joining a group, and given validated survey questions about managing emotions and social interactions since injury. Current or past participation in peer support was low (4.2%), and 30.3% of patients not already in support group were interested in joining. Interest in future participation in peer support was highest among Hispanic patients (37.0%) and lowest among Black patients (0%). Logistic regression models demonstrated that increased total body surface area burned, hospital length of stay, and need for surgical intervention were associated with interest in joining or having joined a peer support group. Effectiveness of management of emotions and social interactions were not associated with interest in joining peer support in the future. These findings demonstrate a considerable difference between levels of interest and participation in peer support within this population. Improving access to and education about benefits of peer support in underresourced communities may help to address the variation in psychosocial outcomes of patients across racial or ethnic minority groups recovering from burns.

US hospital webpages on atrial fibrillation ablation: A potential source for misinformation.

INTRODUCTION: Atrial fibrillation (AF) ablation is performed worldwide. To attract patients, hospitals frequently have webpages that tout the success of the procedure. The information disseminated to the public via these webpages has not been systematically reviewed. Our objective was to assess accuracy of information delivered to the public on hospital websites in regard to atrial AF ablation. METHODS: From July 2019 to January 2020, we performed a Google search for all US hospitals registered with Medicare to see if they had a webpage describing AF ablation. Resulting hospital webpages were abstracted for data on AF ablation success rates and risks. Success rates over 86%, the highest success rate in the medical literature, were deemed exaggerated. RESULTS: Among 4805 hospitals, 487 had webpages describing AF ablation and 33 discussed success rates of AF ablation. Twelve percentage reported exaggerated success rates, 3% referred to ablation as a cure, and 2.8% referred to ablation as a tool to eliminate AF. Less than 10% of webpages describing AF ablation noted the potential need for a second ablation to achieve the stated success rate and merely 16% mentioned risks of the procedure. One percentage of webpages directly suggested AF ablation could reduce risk of stroke while others indirectly suggested it by discussing cessation of anticoagulation. Two webpages mentioned reduced mortality. CONCLUSION: US hospital webpages rarely discuss AF ablation. When discussed, there were concerning unsubstantiated claims regarding mortality, stroke prevention, and need for medical therapy. This could lead to some patients undergoing AF ablation based on faulty understanding.

Comparisons of α2-Adrenergic Agents, Medetomidine and Xylazine, with Pentobarbital for Anesthesia: Important Pitfalls in Diabetic and Nondiabetic Rats.

Purpose: Anesthesia is necessary to conduct rodent electroretinograms (ERGs). We evaluated utility of the α2-agonist medetomidine versus xylazine for ERG studies in nondiabetic and diabetic rats. Pentobarbital was included as a comparator. Methods: Male Sprague-Dawley rats, with and without streptozotocin (STZ)-induced diabetes, were anesthetized with medetomidine (1 mg/kg), xylazine (10 mg/kg) (both with ketamine 75 mg/kg), or pentobarbital (70 mg/kg). The depth of anesthesia was assessed, and if adequate, scotopic ERGs were recorded. Blood glucose was monitored. Results: In nondiabetic rats, all three agents induced satisfactory anesthesia, but with differing durations: medetomidine > pentobarbital > xylazine. ERG responses were similar under medetomidine and xylazine, but relatively reduced under pentobarbital. Both α2-agonists (but not pentobarbital) elicited marked hyperglycemia (peak values 316.1 ± 42.6 and 300.3 ± 29.5 mg/dL, respectively), persisting for 12 h. In diabetic rats, elevated blood glucose concentrations were not affected by any of the agents, but the depth of anesthesia under medetomidine and xylazine was inadequate for ERG recording. Conclusions: In nondiabetic rats, medetomidine and xylazine elicited comparable effects on ERGs that differ from pentobarbital, but both perturbed glucose metabolism, potentially confounding experimental outcomes. In STZ-diabetic rats, neither α2-agent provided adequate anesthesia, while pentobarbital did so. Problems with α2-anesthetic agents, including medetomidine, must be recognized to ensure meaningful interpretation of experimental results.

Length of Cervical Stenosis, Admission ASIA Motor Scores, and BASIC Scores Are Predictors of Recovery Rate Following Central Cord Syndrome.

STUDY DESIGN: A retrospective cohort study. OBJECTIVE: This study aims to determine whether quantitative magnetic resonance imaging (MRI) parameters and radiological scoring systems could be used as a reliable assessment tool for predicting neurological recovery trajectory following acute traumatic central cord injury syndrome (CCS). SUMMARY OF BACKGROUND DATA: Controversy remains in whether CCS should be managed conservatively or by early surgical decompression. It is essential to understand how clinical and radiological parameters correlate with neurological deficits and how they predict recovery trajectories. METHODS: We identified patients with CCS admitted between 2011 and 2018 with a minimum of 1-year follow-up. Cervical MRIs were analyzed for cord/canal dimensions, Brain and Spinal Injury Center (BASIC) scores and sagittal grading as ordinal scales of intraparenchymal cord injury. Japanese Orthopaedic Association (JOA) recovery rates (≥50% as good, < 50% as poor) were analyzed against these variables by logistic regression and receiver operator characteristic (ROC) curves. Additionally, we evaluated American Spinal Injury Association motor scale (AMS) scores/recovery rates. RESULTS: Sixty patients were included, of which 30 were managed conservatively and 30 via surgical decompression. The average follow-up duration for the entire cohort was (51.1 ±â€Š25.7) months. Upon admission, sagittal grading correlated with AMS and JOA scores (P < 0.01, ß = 0.48). Volume of the C2 to C7 canal and axial cord area over the site of maximal compression correlated with AMS and JOA scores respectively (P = 0.04, ß = 0.26; P = 0.01, ß = 0.28). We determined admission AMS more than 61 to be a clinical cutoff for good recovery (area under the receiver operating curve [AUC] = 0.74, 95% confidence interval [CI]: 0.61-0.85, sensitivity 80.9%, specificity 69.2%, P < 0.01). Radiological cutoffs to identify patients with poor recovery rates were length of cervical spinal stenosis more than 3.9 cm (AUC = 0.76, 95% CI: 0.63-0.87, specificity 91.7%, sensitivity 52.2%, P < 0.01), BASIC score of more than 1 (AUC = 0.69, 95% CI: 0.56-0.81, specificity 80.5%, sensitivity 51.1%, P = 0.02). Surgical decompression performed as a salvage procedure upon plateau of recovery did not improve neurological outcomes. CONCLUSION: Clinical and radiological parameters upon presentation were prognosticative of neurological recovery rates in CCS. Surgery performed beyond the acute post-injury period failed to improve outcomes.Level of Evidence: 3.

Chronic cannabis smoking-enriched oral pathobiont drives behavioral changes, macrophage infiltration, and increases ß-amyloid protein production in the brain.

BACKGROUND: Little is known about chronic cannabis smoking-associated oral microbiome and its effects on central nervous system (CNS) functions. METHODS: In the current study, we have analyzed the saliva microbiome in individuals who chronically smoked cannabis with cannabis use disorder (n = 16) and in non-smoking controls (n = 27). The saliva microbiome was analyzed using microbial 16S rRNA sequencing. To investigate the function of cannabis use-associated oral microbiome, mice were orally inoculated with live Actinomyces meyeri, Actinomyces odontolyticus, or Neisseria elongata twice per week for six months, which mimicked human conditions. FINDINGS: We found that cannabis smoking in humans was associated with oral microbial dysbiosis. The most increased oral bacteria were Streptococcus and Actinomyces genus and the most decreased bacteria were Neisseria genus in chronic cannabis smokers compared to those in non-smokers. Among the distinct species bacteria in cannabis smokers, the enrichment of Actinomyces meyeri was inversely associated with the age of first cannabis smoking. Strikingly, oral exposure of Actinomyces meyeri, an oral pathobiont, but not the other two control bacteria, decreased global activity, increased macrophage infiltration, and increased ß-amyloid 42 protein production in the mouse brains. INTERPRETATION: This is the first study to reveal that long-term oral cannabis exposure is associated oral enrichment of Actinomyces meyeri and its contributions to CNS abnormalities.

Modelling preeclampsia: a comparative analysis of the common human trophoblast cell lines.

Preeclampsia remains a challenge without an effective therapy. Evidence supports targetability of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), which are released excessively from the placenta under ischemic and hypoxic stresses. We compared four trophoblast cell lines, BeWo, Jar, Jeg-3, and HTR-8/SVneo, in order to identify a suitable model for drug screening. Cultured trophoblasts were exposed to 1% oxygen vs. normoxia for 24-48 hr; human umbilical vein and aortic endothelial cells were included for comparison. Supernatant sFlt-1 and sEng concentrations were measured by ELISA, and sFlt-1 mRNA expression determined by RT-PCR. Cellular responses to experimental therapeutics were explored. All four trophoblast lines secreted sEng, which did not increase by hypoxia. BeWo, Jar, and Jeg-3 exhibited significantly enhanced expression of sFlt-1 i13 and e15a mRNA in response to hypoxia; however, only BeWo released a detectable level of sFlt-1 protein, which was doubled by hypoxia. In contrast, hypoxia decreased sFlt-1 mRNA expression and protein release in HTR-8/SVneo, similarly to endothelial cells. The cellular mechanism involved HIFα. BeWo responded to representative agents similarly to human primary placental tissues in the literature. These data support that the BeWo-hypoxia model mimics a key pathogenic mechanism of preeclampsia and has potential value for translational drug discovery.

Effects of modified lipoproteins on human trophoblast cells: a role in pre-eclampsia in pregnancies complicated by diabetes.

INTRODUCTION: Pre-eclampsia (PE) is increased ~4-fold by maternal diabetes. Elevated plasma antiangiogenic factors, soluble fms-like tyrosine kinase (sFLT-1) and soluble endoglin (sENG), precede PE onset. We investigated whether diabetes-related stresses, modified lipoproteins and elevated glucose enhance trophoblast sFLT-1 and sENG release and/or alter placental barrier function and whether oxidized low-density lipoprotein (Ox-LDL) is in placental tissue. RESEARCH DESIGN AND METHODS: HTR8/SVneo cells were exposed to 'heavily-oxidized, glycated' LDL (HOG-LDL) versus native LDL (N-LDL) (10-200 mg protein/L) for 24 hours ±pretreatment with glucose (30 mmol/L, 72 hours). Concentrations of sFLT-1 and sENG in supernatants (by ELISA) and expressions of sFLT-1-I13 and sFLT-1-E15A isoforms, endoglin (ENG) and matrix metalloproteinase-14 (MMP-14; by RT-PCR) were quantified. For barrier studies, JAR cells were cultured in Transwell plates (12-14 days), then exposed to LDL. Transepithelial electrical resistance (TEER) was measured after 6, 12 and 24 hours. In placental sections from women with and without type 1 diabetes, immunostaining of apolipoprotein B100 (ApoB, a marker of LDL), Ox-LDL and lipoxidation product 4-hydroxynonenal was performed. RESULTS: HOG-LDL (50 mg/L) increased sFLT-1 (2.7-fold, p<0.01) and sENG (6.4-fold, p<0.001) in supernatants versus N-LDL. HOG-LDL increased expression of sFLT-1-I13 (twofold, p<0.05), sFLT-1-E15A (1.9-fold, p<0.05), ENG (1.6-fold, p<0.01) and MMP-14 (1.8-fold, p<0.05) versus N-LDL. High glucose did not by itself alter sFLT-1 or sENG concentrations, but potentiated effects of HOG-LDL on sFLT-1 by 1.5-fold (p<0.05) and on sENG by 1.8-fold (p<0.01). HOG-LDL (200 mg/L) induced trophoblast barrier impairment, decreasing TEER at 6 hours (p<0.01), 12 hours (p<0.01) and 24 hours (p<0.05) versus N-LDL. Immunostaining of term placental samples from women both with and without diabetes revealed presence of intravillous modified lipoproteins. CONCLUSION: These findings may explain, in part, the high risk for PE in women with diabetes. The trophoblast culture model has potential for evaluating novel therapies targeting barrier dysfunction.

Differential regulation of a placental SAM68 and sFLT1 gene pathway and the relevance to maternal vitamin D sufficiency.

OBJECTIVE: The goal of this study was to determine if an axis of placental gene expression associated with early onset and severe preeclampsia (EOSPE) was operative in term pregnancy and correlated with vitamin D sufficiency. METHODS: qPCR analysis of NKX2-5, SAM68, sFLT1 and membrane bound VEGFR1/FLT1 mRNA expression was conducted in placentas from 43 subjects enrolled in a vitamin D3 pregnancy supplementation trial. Pair-wise rank order correlations between patient-specific gene expression levels were calculated, and their relationship to maternal 25(OH)D status was assessed by a two-sample Wilcoxon test. Additionally, we probed the mechanistic link between SAM68 and sFLT1 using siRNA depletion in a human trophoblast cell line model. RESULTS: Positive and highly significant correlations were found between SAM68 vs. sFLT1 and SAM68 vs. FLT1 expression levels, as were significant and differential correlations between the expression of these genes and perinatal 25(OH)D status. The variability when stratified by race/ethnicity was qualitatively distinct from those previously observed in EOSPE. Mechanistic studies confirmed a functional role for SAM68 protein in the regulation of sFLT1 expression. NKX2-5 expression was not significantly correlated with sFLT1 or SAM68 expression in these samples, suggesting that its expression may be significant at earlier stages of pregnancy or be restricted to pathological settings. CONCLUSIONS: These data further support our overarching hypothesis that SAM68 expression is a key determinant of VEGFR1 isoform expression in the placenta, and provide additional insights into how this gene pathway may be differentially deployed or modified in normal and pathological pregnancies.

Effects of Modified Low-Density Lipoproteins and Fenofibrate on an Outer Blood-Retina Barrier Model: Implications for Diabetic Retinopathy.

Purpose: There is a lack of treatment for early diabetic retinopathy (DR), including blood-retina barrier (BRB) breakdown. The robust clinical benefit of fenofibrate in DR provides an opportunity to explore disease mechanisms and therapeutic targets. We have previously found that modified lipoproteins contribute to DR and that fenofibrate protects the inner BRB. We now investigate (1) whether modified lipoproteins elicit outer BRB injury and (2) whether fenofibrate may alleviate such damage. Methods: Human retinal pigment epithelium ARPE-19 cells were cultured in semipermeable transwells to establish a monolayer barrier and then exposed to heavily oxidized, glycated low-density lipoprotein (HOG-LDL, 25-300 mg/L, up to 24 h) versus native (N)-LDL. Transepithelial electric resistance (TEER) and FITC-dextran permeability were measured. The effects of fenofibrate, its active metabolite fenofibric acid, and other peroxisome proliferator-activated receptor (PPARα) agonists (gemfibrozil, bezafibrate, and WY14643) were evaluated, with and without the PPARα antagonist GW6471 or the adenosine monophosphate-activated protein kinase (AMPK) inhibitor Compound C. Results: HOG-LDL induced concentration- and time-dependent barrier impairment, decreasing TEER and increasing dextran leakage, effects that were amplified by high glucose. Fenofibric acid, but not fenofibrate, gemfibrozil, bezafibrate, or WY14643, attenuated barrier impairment. This effect was reversed significantly by Compound C, but not by GW6471. Conclusions: Modified lipoproteins elicited outer BRB injury in an experimental model, which was reduced by fenofibric acid through a PPARα-independent, AMPK-mediated mechanism. These findings suggest a protective role of fenofibric acid on the outer BRB in diabetic retina.

Vitamin D Metabolites and Binding Protein Predict Preeclampsia in Women with Type 1 Diabetes.

The risk for preeclampsia (PE) is enhanced ~4-fold by the presence of maternal type 1 diabetes (T1DM). Vitamin D is essential for healthy pregnancy. We assessed the total, bioavailable, and free concentrations of plasma 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), and vitamin D binding protein (VDBP) at ~12, ~22, and ~32 weeks' gestation ("Visits" (V) 1, 2, and 3, respectively) in 23 T1DM women who developed PE, 24 who remained normotensive, and 19 non-diabetic, normotensive women (reference controls). 25(OH)D deficiency was more frequent in diabetic than non-diabetic women (69% vs. 22%, p < 0.05), but no measure of 25(OH)D predicted PE. By contrast, higher 1,25(OH)2D concentrations at V2 (total, bioavailable, and free: p < 0.01) and V3 (bioavailable: p < 0.05; free: p < 0.01), lower concentrations of VDBP at V3 (p < 0.05), and elevated ratios of 1,25(OH)2D/VDBP (V2, V3: p < 0.01) and 1,25(OH)2D/25(OH)D (V3, p < 0.05) were all associated with PE, and significance persisted in multivariate analyses. In summary, in women with T1DM, concentrations of 1,25(OH)2D were higher, and VDBP lower, in the second and third trimesters in women who later developed PE than in those who did not. 1,25(OH)2D may serve as a new marker for PE risk and could be implicated in pathogenesis.