Failed Extubation in Neonates After Cardiac Surgery: A Single-Center, Retrospective Study.

OBJECTIVES: To describe factors associated with failed extubation (FE) in neonates following cardiovascular surgery, and the relationship with clinical outcomes. DESIGN: Retrospective cohort study. SETTING: Twenty-bed pediatric cardiac ICU (PCICU) in an academic tertiary care children's hospital. PATIENTS: Neonates admitted to the PCICU following cardiac surgery between July 2015 and June 2018. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients who experienced FE were compared with patients who were successfully extubated. Variables associated with FE ( p < 0.05) from univariate analysis were considered for inclusion in multivariable logistic regression. Univariate associations of FE with clinical outcomes were also examined. Of 240 patients, 40 (17%) experienced FE. Univariate analyses revealed associations of FE with upper airway (UA) abnormality (25% vs 8%, p = 0.003) and delayed sternal closure (50% vs 24%, p = 0.001). There were weaker associations of FE with hypoplastic left heart syndrome (25% vs 13%, p = 0.04), postoperative ventilation greater than 7 days (33% vs 15%, p = 0.01), Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery (STAT) category 5 operations (38% vs 21%, p = 0.02), and respiratory rate during spontaneous breathing trial (median 42 vs 37 breaths/min, p = 0.01). In multivariable analysis, UA abnormalities (adjusted odds ratio [AOR] 3.5; 95% CI, 1.4-9.0), postoperative ventilation greater than 7 days (AOR 2.3; 95% CI, 1.0-5.2), and STAT category 5 operations (AOR 2.4; 95% CI, 1.1-5.2) were independently associated with FE. FE was also associated with unplanned reoperation/reintervention during hospital course (38% vs 22%, p = 0.04), longer hospitalization (median 29 vs 16.5 d, p < 0.0001), and in-hospital mortality (13% vs 3%, p = 0.02). CONCLUSIONS: FE in neonates occurs relatively commonly following cardiac surgery and is associated with adverse clinical outcomes. Additional data are needed to further optimize periextubation decision-making in patients with multiple clinical factors associated with FE.

Methods to Enhance Causal Inference for Assessing Impact of Clinical Informatics Platform Implementation.

BACKGROUND: Hospitals are increasingly likely to implement clinical informatics tools to improve quality of care, necessitating rigorous approaches to evaluate effectiveness. We leveraged a multi-institutional data repository and applied causal inference methods to assess implementation of a commercial data visualization software in our pediatric cardiac intensive care unit. METHODS: Natural experiment in the University of Michigan (UM) Cardiac Intensive Care Unit pre and postimplementation of data visualization software analyzed within the Pediatric Cardiac Critical Care Consortium clinical registry; we identified N=21 control hospitals that contributed contemporaneous registry data during the study period. We used the platform during multiple daily rounds to visualize clinical data trends. We evaluated outcomes-case-mix adjusted postoperative mortality, cardiac arrest and unplanned readmission rates, and postoperative length of stay-most likely impacted by this change. There were no quality improvement initiatives focused specifically on these outcomes nor any organizational changes at UM in either era. We performed a difference-in-differences analysis to compare changes in UM outcomes to those at control hospitals across the pre versus postimplementation eras. RESULTS: We compared 1436 pre versus 779 postimplementation admissions at UM to 19 854 (pre) versus 14 160 (post) at controls. Admission characteristics were similar between eras. Postimplementation at UM we observed relative reductions in cardiac arrests among medical admissions, unplanned readmissions, and postoperative length of stay by -14%, -41%, and -18%, respectively. The difference-in-differences estimate for each outcome was statistically significant (P<0.05), suggesting the difference in outcomes at UM pre versus postimplementation is statistically significantly different from control hospitals during the same time. CONCLUSIONS: Clinical registries provide opportunities to thoroughly evaluate implementation of new informatics tools at single institutions. Borrowing strength from multi-institutional data and drawing ideas from causal inference, our analysis solidified greater belief in the effectiveness of this software across our institution.

Data-driven computational models of ventricular-arterial hemodynamics in pediatric pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a complex disease involving increased resistance in the pulmonary arteries and subsequent right ventricular (RV) remodeling. Ventricular-arterial interactions are fundamental to PAH pathophysiology but are rarely captured in computational models. It is important to identify metrics that capture and quantify these interactions to inform our understanding of this disease as well as potentially facilitate patient stratification. Towards this end, we developed and calibrated two multi-scale high-resolution closed-loop computational models using open-source software: a high-resolution arterial model implemented using CRIMSON, and a high-resolution ventricular model implemented using FEniCS. Models were constructed with clinical data including non-invasive imaging and invasive hemodynamic measurements from a cohort of pediatric PAH patients. A contribution of this work is the discussion of inconsistencies in anatomical and hemodynamic data routinely acquired in PAH patients. We proposed and implemented strategies to mitigate these inconsistencies, and subsequently use this data to inform and calibrate computational models of the ventricles and large arteries. Computational models based on adjusted clinical data were calibrated until the simulated results for the high-resolution arterial models matched within 10% of adjusted data consisting of pressure and flow, whereas the high-resolution ventricular models were calibrated until simulation results matched adjusted data of volume and pressure waveforms within 10%. A statistical analysis was performed to correlate numerous data-derived and model-derived metrics with clinically assessed disease severity. Several model-derived metrics were strongly correlated with clinically assessed disease severity, suggesting that computational models may aid in assessing PAH severity.

Preventing Cardiac Arrest in the Pediatric Cardiac Intensive Care Unit Through Multicenter Collaboration.

Importance: Preventing in-hospital cardiac arrest (IHCA) likely represents an effective strategy to improve outcomes for critically ill patients, but feasibility of IHCA prevention remains unclear. Objective: To determine whether a low-technology cardiac arrest prevention (CAP) practice bundle decreases IHCA rate. Design, Setting, and Participants: Pediatric cardiac intensive care unit (CICU) teams from the Pediatric Cardiac Critical Care Consortium (PC4) formed a collaborative learning network to implement the CAP bundle consistent with the Institute for Healthcare Improvement framework; 15 hospitals implemented the bundle voluntarily. Risk-adjusted IHCA incidence rates were analyzed across 2 time periods, 12 months (baseline) and 18 months after CAP implementation (intervention) using difference-in-differences (DID) regression to compare 15 CAP and 16 control PC4 hospitals that chose not to participate in CAP but had IHCA rates tracked in the PC4 registry. Patients deemed at high risk for IHCA, based on a priori evidence-based criteria and empirical hospital-specific criteria, were selected to receive the CAP bundle. Data were collected from July 2018 to December 2019, and data were analyzed from March to August 2020. Interventions: CAP bundle included 5 elements developed to promote increased situational awareness and communication among bedside clinicians to recognize and mitigate deterioration in high-risk patients. Main Outcomes and Measures: Risk-adjusted IHCA incidence rate across all CICU admissions (IHCA events divided by all admissions). Results: The bundle was activated in 2664 of 10 510 CAP hospital admissions (25.3%); admission characteristics were similar across study periods. There was a 30% relative reduction in risk-adjusted IHCA incidence rate at CAP hospitals (intervention period: 2.6%; 95% CI, 2.2-2.9; baseline: 3.7%; 95% CI, 3.1-4.0), but no change at control hospitals (intervention period: 2.7%; 95% CI, 2.3-2.9; baseline: 2.7%; 95% CI, 2.2-3.0). DID analysis confirmed significantly reduced odds of IHCA among all admissions at CAP hospitals compared with control hospitals during the intervention period vs baseline (odds ratio, 0.72; 95% CI, 0.56-0.91; P = .01). DID odds ratios were 0.72 (95% CI, 0.53-0.98) for the surgical subgroup, 0.74 (95% CI, 0.48-1.14) for the medical subgroup, and 0.72 (95% CI, 0.50-1.03) for the high-risk admission subgroup at CAP hospitals after intervention. All-cause risk-adjusted mortality rate did not change after intervention. Conclusions and Relevance: Implementation of this CAP bundle led to significant IHCA reduction across multiple pediatric CICUs. Future studies may determine if this bundle can be effective in other critically ill populations.

Critical Care Unit Organizational and Personnel Factors Impact Cardiac Arrest Prevention and Rescue in the Pediatric Cardiac Population.

OBJECTIVES: Patient-level factors related to cardiac arrest in the pediatric cardiac population are well understood but may be unmodifiable. The impact of cardiac ICU organizational and personnel factors on cardiac arrest rates and outcomes remains unknown. We sought to better understand the association between these potentially modifiable organizational and personnel factors on cardiac arrest prevention and rescue. DESIGN: Retrospective analysis of the Pediatric Cardiac Critical Care Consortium registry. SETTING: Pediatric cardiac ICUs. PATIENTS: All cardiac ICU admissions were evaluated for cardiac arrest and survival outcomes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Successful prevention was defined as the proportion of admissions with no cardiac arrest (inverse of cardiac arrest incidence). Rescue was the proportion of patients surviving to cardiac ICU discharge after cardiac arrest. Cardiac ICU organizational and personnel factors were captured via site questionnaires. The associations between organizational and personnel factors and prevention/rescue were analyzed using Fine-Gray and multinomial regression, respectively, accounting for clustering within hospitals. We analyzed 54,521 cardiac ICU admissions (29 hospitals) with 1,398 cardiac arrest events (2.5%) between August 1, 2014, and March 5, 2019. For both surgical and medical admissions, lower average daily cardiac ICU occupancy was associated with better cardiac arrest prevention. Better rescue for medical admissions was observed for higher registered nursing hours per patient day and lower proportions of "part time" cardiac ICU physician staff (< 6 service weeks/yr). Increased registered nurse experience was associated with better rescue for surgical admissions. Increased proportion of critical care certified nurses, full-time intensivists with critical care fellowship training, dedicated respiratory therapists, quality/safety resources, and annual cardiac ICU admission volume were not associated with improved prevention or rescue. CONCLUSIONS: Our multi-institutional analysis identified cardiac ICU bed occupancy, registered nurse experience, and physician staffing as potentially important factors associated with cardiac arrest prevention and rescue. Recognizing the limitations of measuring these variables cross-sectionally, additional studies are needed to further investigate these organizational and personnel factors, their interrelationships, and how hospitals can modify structure to improve cardiac arrest outcomes.

Infundibular sparing versus transinfundibular approach to the repair of tetralogy of Fallot.

INTRODUCTION: The right ventricular infundibular sparing approach (RVIS) to the repair of tetralogy of Fallot (TOF) avoids a full-thickness ventricular incision, typically utilized in the transinfundibular (TI) method. METHODS: We performed a retrospective, age-matched cohort study of patients who underwent RVIS at Texas Children's Hospital or TI at Children's Hospital Medical Center in Nebraska and subsequently underwent cardiac magnetic resonance imaging (CMR). We compared right ventricular end-diastolic and systolic volumes indexed to body surface area (RVEDVi and RVESVi) and right ventricular ejection fraction (RVEF) as primary endpoints. Secondary endpoints were indexed left ventricular diastolic and systolic volume (LVEDVi and LVESVi), left ventricular ejection fraction (LVEF), right ventricular (RV) sinus ejection fraction (EF) and RV outflow tract EF (RVOT EF). RESULTS: Seventy-nine patients were included in the analysis; 40 underwent RVIS and 39 underwent TI repair. None of the patients in the TI repair group had an initial palliation with a systemic to pulmonary arterial shunt compared to seven (18%) in the RVIS group (P < .01). There was no appreciable difference in RVEDVi (122 ± 29 cc/m2 vs 130 ± 29 cc/m2 , P = .59) or pulmonary regurgitant fraction (40 ± 13 vs 37 ± 18, P = .29) between the RVIS and TI groups. Compared to the TI group, the RVIS group had higher RVEF (54 ± 6% vs 44 ± 9%, P < .01), lower RVESV (57 ± 17 cc/m2 vs 67 ± 25 cc/m2 , P = .03), higher LVEF (61 ± 11% vs 54 ± 8%, P < .01), higher RVOT EF (47 ± 12% vs 41 ± 11%, P = .03), and higher RV sinus EF (56 ± 5% vs 49 ± 6%, P < .01) CONCLUSIONS: In this selected cohort, patients who underwent RVIS repair for TOF had higher right and left ventricular ejection fraction compared to those who underwent TI repair.

Current monitoring and innovative predictive modeling to improve care in the pediatric cardiac intensive care unit.

The objectives of this review are (I) to describe the challenges associated with monitoring patients in the pediatric cardiac intensive care unit (PCICU) and (II) to discuss the use of innovative statistical and artificial intelligence (AI) software programs to attempt to predict significant clinical events. Patients cared for in the PCICU are clinically fragile and at risk for fatal decompensation. Current monitoring modalities are often ineffective, sometimes inaccurate, and fail to detect a deteriorating clinical status in a timely manner. Predictive models created by AI and machine learning may lead to earlier detection of patients at risk for clinical decompensation and thereby improve care for critically ill pediatric cardiac patients.

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

OBJECTIVE: Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. METHODS: Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. RESULTS: Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 µg/mg vs. 19.3 ± 5.6 µg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). CONCLUSIONS: A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.

In vivo experiments reveal the good, the bad and the ugly faces of sFlt-1 in pregnancy.

OBJECTIVE: Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia. METHODS: Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy. RESULTS: Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4 × 10(-5)). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice. CONCLUSIONS: Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.

Systemic effects of intracoronary nitroglycerin during coronary angiography in children after heart transplantation.

Coronary spasm during coronary angiography for vasculopathy in children can be prevented by the intracoronary administration of nitroglycerin. We reviewed the anesthesia and catheterization reports and charts for pediatric transplant recipients who underwent angiography from 2005 through 2010. Correlation analysis was used to study the relation of post-injection systolic blood pressure (SBP) to nitroglycerin dose. Forty-one angiographic evaluations were performed on 25 patients (13 male and 12 female). Mean age was 9.9 ± 3.2 years (range, 3.3-16.1 yr). The mean total dose of nitroglycerin was 2.93 ± 1.60 µg/kg (range, 1-8 µg/kg). There was a significant drop between the baseline SBP (mean, 106 ± 21.6 mmHg) and the lowest mean SBP before nitroglycerin administration (78 ± 13.2, P <0.0001, paired t test). There was no significant additional change in SBP (mean after nitroglycerin administration, 80.7 ± 13.1 mmHg; P = 0.2). There was a significant drop in lowest heart rate between baseline (109 ± 16.5 beats/min) and before nitroglycerin administration (89 ± 14.3 beats/min; P <0.0001, paired t test). There was no significant additional change in heart rate (mean heart rate after nitroglycerin, 84 ± 17.7 beats/min; P = 0.09). There were 2 interventions for SBP before nitroglycerin and 2 after nitroglycerin. One child experienced a transient ST-T-segment change during angiography after nitroglycerin. In the highest dose range, the additional decrease in SBP was 7.2 mmHg (P=0.03). Routine intracoronary nitroglycerin administration in this dose range produced no significant changes in SBP or heart rate in children.

MAME models for 4D live-cell imaging of tumor: microenvironment interactions that impact malignant progression.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

Cathepsin B inhibition limits bone metastasis in breast cancer.

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Identification and functional impact of homo-oligomers of the human proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His(10) epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a "dominant-positive" phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.

Maspin reprograms the gene expression profile of prostate carcinoma cells for differentiation.

Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFß signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.

Chiral porphyrazine near-IR optical imaging agent exhibiting preferential tumor accumulation.

A chiral porphyrazine (pz), H(2)[pz(trans-A(2)B(2))] (247), has been prepared that exhibits preferential in vivo accumulation in the cells of tumors. Pz 247 exhibits near-infrared (NIR) emission with lambda > 700 nm in the required wavelength range for maximum tissue penetration. When MDA-MB-231 breast tumor cells are treated with 247, the agent shows strong intracellular fluorescence with an emission maximum, 704 nm, which indicates that it localizes within a hydrophobic microenvironment. Pz 247 is shown to associate with the lipophilic core of LDL and undergo cellular entry primarily through receptor-mediated endocytosis accumulating in lysosomes. Preliminary in vivo studies show that 247 exhibits preferential accumulation and retention in the cells of MDA-MB-231 tumors subcutaneously implanted in mice, thereby enabling NIR optical imaging with excellent contrast between tumor and surrounding tissue. The intensity of fluorescence from 247 within the tumor increases over time up to 48 h after injection presumably due to the sequestration of circulating 247/LDL complex by the tumor tissue. As the need for cholesterol, and thus LDL, is elevated in highly proliferative tumor cells over nontumorigenic cells, 247 has potential application for all such tumors.

Bone marrow-derived cathepsin K cleaves SPARC in bone metastasis.

Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis.

Imaging and quantifying the dynamics of tumor-associated proteolysis.

The roles of proteases in cancer are dynamic. Furthermore, the roles or functions of any one protease may differ from one stage of cancer to another. Proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as from tumor cells make important contributions to 'tumor proteolysis'. Many tumors exhibit increases in expression of proteases at the level of transcripts and protein; however, whether those proteases play causal roles in malignant progression is known for only a handful of proteases. What the critical substrate or substrates that are cleaved in vivo by any given protease is also known for only a few proteases. Therefore, the recent development of techniques and reagents for live cell imaging of protease activity, in conjunction with informed knowledge of critical natural substrates, should help to define protease functions. Here we describe live cell assays for imaging proteolysis, protocols for quantifying proteolysis and the use of such assays to follow the dynamics of proteolysis by tumor cells alone and tumor cells interacting with other cells found in the tumor microenvironment. In addition, we describe an in vitro model that recapitulates the architecture of the mammary gland, a model designed to determine the effects of dynamic interactions with the surrounding microenvironment on 'tumor proteolysis' and the respective contributions of various cell types to 'tumor proteolysis'. The assays and models described here could serve as screening platforms for the identification of proteolytic pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use.

Visualizing protease activity in living cells: from two dimensions to four dimensions.

Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal microscopy and advanced imaging software, the fluorescence is detected and accurate measurements of proteolytic degradation in three and four dimensions can be assessed.

Pre- and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex.

The NMDA receptor is one of the ionotropic glutamate receptors essential for excitatory neurotransmission and synaptic plasticity. The present study examined the ontogenies of NMDAR1 and NMDAR2B during development (embryonic days: (E) 14, 15, 16, 17 and postnatal days: (P) 0, 5, 10, 20, and 60). The immunoreactivities of both NMDAR1 and NMDAR2B were visualized and semi- quantified simultaneously using batch-matched procedures during the developmental period. In addition, the heteromeric coassembly of NMDAR1 (NR1) and NMDAR2B (NR2B) was analyzed throughout development using immunocoprecipitation blots. Differential developmental expressions were observed between the two NMDA receptor subunits. Although both NR1 and NR2B were already expressed on cortical neurons at E14, NR1 immunoreactivity was lower than NR2B but increased up to P20 then was lower at the last sampling day (P60). By comparison, the high NR2B immunoreactivity at E14 increased until P5, and until the next sample day (P60). Despite the early embryonic protein expressions in prenatal cortex both NR1 and NR2B immunocoprecipitation analyses showed that NR1-NR2B coassembly was not initially detectable until birth (P0), and increased up to P20. This suggests that the physically interactive NMDAR1-2B complex may contribute to the plasticity of early postnatal development characterized by axonal dendritic synaptogenesis.