Citations

Peter J. Psaltis, 2011 Finalist and Presenting Author

Background: Hematopoietic stem cells and the vasculature share a unique relationship during development, in the adult bone marrow and in the setting of extramedullary hematopoiesis within diseased calcified vessels. We hypothesized that the adult vasculature may contain progenitor cells with the capacity to reconstitute multi-lineage hematopoietic cells. Methods: Aortas from normal and ApoE −/− adult mice were disaggregated with collagenase and elastase. Disaggregates were cultured in methylcellulose for quantification of short and long term hematopoietic colony forming units (CFU). Flow cytometry assessed various populations of hematopoietic progenitor cells. Aortic disaggregates (or bone marrow or blood) from ROSA26 and GFP mice were transplanted into sublethally irradiated C57BL/6 recipients (n=6) via tail vein injections. Bone marrow cells from the primary recipients were transplanted at 4 months to secondary C57BL/6 recipients (n=6). Mice were sacrificed 3 months later. Blood and bone marrow were analyzed. Results: In short term culture in methylcellulose, aortic disaggregates generated erythroid, macrophage, granulocyte and megakaryocyte CFU, with predominance of macrophage colonies, primarily from adventitia (p<0.05) and a greater frequency was found in Apo E −/− mice (p<0.05). Long term culture defined that the combined frequency of the hematopoietic precursors approximates the frequency found in bone marrow (1:47,277). Flow cytometry of the aortic disaggregates indicates that among lin-/c-kit+/sca-1+ cells a small population of hematopoietic stem cells (CD150+endoglin+) and a population of multipotent progenitor cells (CD150− endoglin−) exist. Infusion of aortic disaggregates generated bone marrow, blood and spleen chimerism in primary and secondary transplanted mice at 3– 4 months. Flow cytometry of peripheral cells demonstrated multilineage hematopoietic cells. Conclusions: For the first time, we have identified a variety of progenitor cell populations in the aorta capable of hematopoiesis. Infusion of these cells into sublethally irradiated mice results in stable chimeras. These data support the presence of novel aortic hematopoietic progenitor cells.

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P < 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.

The purpose of this study was to evaluate the biodistribution of a platelet-derived exosome product (PEP), previously shown to promote regeneration in the setting of wound healing, in a porcine model delivered through various approaches. Exosomes were labeled with DiR far-red lipophilic dye to track and quantify exosomes in tissue, following delivery via intravenous, pulmonary artery balloon catheter, or nebulization in sus scrofa domestic pigs. Following euthanasia, far-red dye was detected by Xenogen IVUS imaging, while exosomal protein CD63 was detected by Western blot and immunohistochemistry. Nebulization and intravenous delivery both resulted in global uptake of exosomes within the lung parenchyma. However, nebulization resulted in the greatest degree of exosome uptake. Pulmonary artery balloon catheter-guided delivery provided the further ability to localize pulmonary delivery. No off-target absorption was noted in the heart, spleen, or kidney. However, the liver demonstrated uptake primarily in nebulization-treated animals. Nebulization also resulted in uptake in the trachea, without significant absorption in the esophagus. Overall, this study demonstrated the feasibility of pulmonary delivery of exosomes using nebulization or intravenous infusion to accomplish global delivery or pulmonary artery balloon catheter-guided delivery for localized delivery.

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Introduction: Patients with coronary artery disease (CAD) not amenable to revascularization are reported to have a 3-year mortality >14%. Advances in treatment modalities including medical management, rotational atherectomy, balloon lithotripsy, and drug-eluting stents work for focal disease but poorly treat diffuse CAD. In a porcine model of in-stent restenosis, we here evaluated whether a pro-angiogenic platelet-derived exosome product (PEP) could be delivered into the perivascular space to foster collateral vessel formation. Methods/Results: Cobalt chromium stents were placed and were oversized by 1 mm in the left anterior descending (LAD) and posterior descending arteries (PDA) of sus scrofa domesticus pigs. In-stent restenosis was angiographically visualized at the 2-month timepoint, with hemodynamic significance documented with fractional flow reserve (FFR) of <0.8. To document feasibility of PEP delivery into the perivascular space, exosomes were labeled with a far-red fluorescent tag and delivered into the perivascular bed. Here, the Bullfrog® micro-infusion catheter was positioned either in the great cardiac vein or the middle cardiac vein and PEP was delivered with the microneedle pointed towards the LAD or the PDA. Biodistribution demonstrated exosome retention and uptake around cardiac veins. Using the Bullfrog® technique, PEP was delivered in the perivascular space adjacent to areas of significant coronary stenosis. Initial results 1-month post-treatment demonstrated improved FFR of treated stenotic vessels (from 0.68 to 0.85), while untreated vessels demonstrate no change from previous FFR measurement (from 0.79 to 0.78). Conclusions: Here, exosome-mediated angiogenesis in the perivascular space improved coronary blood flow, distal to areas of stenosis. In patients with diffuse CAD, delivery of biotherapy in cardiac veins running anatomically parallel to stenosed epicardial arteries provides a novel therapeutic opportunity to supplement the existing armamentarium of therapies available for in-stent restenosis.

Introduction: At the forefront of post-heart transplant care is early detection of transplant rejection. However, the current gold standard for detecting acute cellular transplant rejection is recurrent invasive endomyocardial biopsies (EMB). We aimed at developing a non-invasive biomarker for acute cellular heart transplant rejection (ACR) using a plasma extracellular vesicle (EV) platform. Methods: Peripheral and coronary sinus plasma samples were prospectively collected from 183 (31.7% female) patients from 2020 to 2023 during routine surveillance EMB. A discovery cohort comprised five patients who displayed ISHLT 0-1R biopsy followed by an ISHLT 2R grade rejection event. Plasma EVs were isolated from the baseline (0-1R) and rejection (2R) time points. Subsequently, integrative analysis of plasma EV proteomic, miRNomic, and metabolomics was performed pairwise with the samples. Results: The study collected 943 sample time points from 183 patients. Of these sample collections, 43 (4.5%) cases were 2R rejections. Our proteomic analysis of the plasma EVs identified 28 proteins found explicitly during a grade 2R rejection and 87 differentially expressed proteins during a 2R rejection event. An Ingenuity Pathway Analysis (IPA) analysis of the upregulated proteins highlighted EVs mediated mTOR signaling and antigen presentation as central to rejection events. Although all patients in the discovery cohort were negative for antibody-mediated rejection (AMR), the proteomic findings also revealed increased B-cell receptor signaling, implying a mechanistic intersection of ACR and AMR. Conclusions: A distinct set of proteins were found in plasma EVs during ACR events, providing potentially specific and sensitive biomarkers to reduce the need for EMB that could be exploited for ACR diagnosis. The proteins identified formed an immune signaling network that suggests a novel role for EVs in ACR pathogenesis.

Coronary angiography is the gold standard for the diagnosis and treatment of epicardial coronary artery disease. Further, it is an essential tool for conduction of translational cardiac research on large animal models, in the development of novel cardiac therapies. While in humans coronary angiography has an exceedingly low mortality rate; in pigs, procedural mortality has been reported as high as 15%, likely due to unsuitable variations in angiographic techniques. Porcine models are used due to the resemblance of cardiac function and anatomy to humans. However, procedural guidelines delineating proper equipment to be used for angiography in this model has yet to be systematically assessed. Aims: To evaluate clinically common catheters for their utility in the safe engagement of the coronary vessels in pigs. Establish optimal workflow and fluoroscopy views for safe coronary artery engagement and angiography. Method/Results: Percutaneous superficial femoral artery access was acquired via ultrasound guidance and subsequent cardiac catheterization was performed in sus domesticus weighing 35-50 kg. Catheters that were tested included the JL 2.5, JL3.0, JR 3.0, AR1, AL1, MPA1, Champ1, 3DRC, hockey stick and RCB (5-7 french) operated over a standard .035 J tip wire. AP views were optimum to engage both the left and right coronary vessels. Only AR1, 3DRC, and hockey stick catheters successfully engaged the RCA, while all catheters tested could successfully engage the LCA. AR1 was noted to be the best for RCA engagement and AL1 for Left system engagement. AR1 was also the most succesful in engagement of both LM and RCA in same pig. AL1 provided the most stable engagement for Left coronary system. AR1/JL were better for LAD intervensions. AL1 is best for LCX intervensions Conclusion: We present a systematic description of percutaneous coronary angiographic procedures highlighting that AL for the LCA and AR for the RCA are the best choice of catheters for fastest and safest engagement of the coronary arteries limiting both radiation time and contrast use. While variations in anatomy can mandate changes in angiographic angles and catheter techniques, AP visualization and use of AL and AR catheters provided the most consistent and effective approach for porcine angiography.

OBJECTIVES Early warning scores detecting clinical deterioration in pediatric inpatients have wide-ranging performance and use a limited number of clinical features. This study developed a machine learning model leveraging multiple static and dynamic clinical features from the electronic health record to predict the composite outcome of unplanned transfer to the ICU within 24 hours and inpatient mortality within 48 hours in hospitalized children. METHODS Using a retrospective development cohort of 17 630 encounters across 10 388 patients, 2 machine learning models (light gradient boosting machine [LGBM] and random forest) were trained on 542 features and compared with our institutional Pediatric Early Warning Score (I-PEWS). RESULTS The LGBM model significantly outperformed I-PEWS based on receiver operating characteristic curve (AUROC) for the composite outcome of ICU transfer or mortality for both internal validation and temporal validation cohorts (AUROC 0.785 95% confidence interval [0.780–0.791] vs 0.708 [0.701–0.715] for temporal validation) as well as lead-time before deterioration events (median 11 hours vs 3 hours; P = .004). However, LGBM performance as evaluated by precision recall curve was lesser in the temporal validation cohort with associated decreased positive predictive value (6% vs 29%) and increased number needed to evaluate (17 vs 3) compared with I-PEWS. CONCLUSIONS Our electronic health record based machine learning model demonstrated improved AUROC and lead-time in predicting clinical deterioration in pediatric inpatients 24 to 48 hours in advance compared with I-PEWS. Further work is needed to optimize model positive predictive value to allow for integration into clinical practice.

Context.— Cytologic-histologic correlation (CHC) is a Clinical Laboratory Improvement Amendments–mandated requirement for gynecologic cytology, but no similar requirement exists for nongynecologic cytology. This study presents the findings from a College of American Pathologists survey of nongynecologic cytology practice patterns. Objective.— To survey the current CHC practices for nongynecologic cytology. Design.— Data were analyzed from a survey developed by the committee and distributed to participants in the Nongynecologic Cytopathology Education Program mailing. Results.— Adoption of CHC for nongynecologic cytology cases is worldwide, with 88.5% of institutions performing CHC on these specimens, a substantial increase from previous years. Performance of CHC varied by institution type, with clinic or regional/local independent laboratories and national/corporate laboratories performing CHC significantly less frequently than hospitals, university hospitals/academic medical centers, and Veterans Administration/Department of Defense hospital institutions. Most CHC was performed concurrently in real time, when the corresponding surgical specimen was reviewed. Selection for real-time concurrent CHC was by the interpreting pathologist, the pathologist diagnosing the surgical biopsy sample or cytopathology case, or both. Sampling was by far the most common reason for discordance. A 2-step difference was the most frequent threshold for discordance between cytology and surgical specimens, but this criterion varied among institutions, with no majority definition. The positive predictive value of a positive cytology finding was calculated rarely in North American institutions but was calculated more frequently in international institutions. Conclusions.— CHC practices for nongynecologic cytopathology mirror those found for CHC of gynecologic cytopathology.