Multiparameter quantitative analyses of diagnostic cells in brain tissues from tuberous sclerosis complex.

The advent of high-dimensional imaging offers new opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example case is found in tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine learning pipeline (BAlloon IDENtifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom 36-antibody panel and imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC marker proteins and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. Here, we present a modular workflow encompassing BAIDEN, a custom antibody panel, a control sample microarray, and analysis pipelines-both open-source and in-house-and apply this workflow to understand the abundance, structure, and signaling activity of BCs as an example case of how high-dimensional imaging can be applied within human tissues.

Management of Hypertension in the Asia-Pacific Region: A Structured Review.

This article reviews available evidence regarding hypertension management in the Asia-Pacific region, focussing on five research questions that deal with specific aspects: blood pressure (BP) control, guideline recommendations, role of renin-angiotensin-aldosterone system (RAAS) inhibitors in clinical practice, pharmacological management and real-world adherence to guideline recommendations. A PubMed search identified 2537 articles, of which 94 were considered relevant. Compared with Europeans, Asians have higher systolic/diastolic/mean arterial BP, with a stronger association between BP and stroke. Calcium channel blockers are the most-commonly prescribed monotherapy in Asia, with significant variability between countries in the rates of angiotensin-converting enzyme inhibitors (ACEis)/angiotensin-receptor blockers (ARBs) and single-pill combination (SPC) use. In clinical practice, ARBs are used more commonly than ACEis, despite the absence of recommendation from guidelines and clinical evidence supporting the use of one class of drug over the other. Ideally, antihypertensive treatment should be tailored to the individual patient, but currently there are limited data on the characteristics of hypertension in Asia-Pacific individuals. Large outcome studies assessing RAAS inhibitor efficacy and safety in multi-national Asian populations are lacking. Among treated patients, BP control rates were ~ 35 to 40%; BP control in Asia-Pacific is suboptimal, and disproportionately so compared with Western nations. Strategies to improve the management of hypertension include wider access/availability of affordable treatments, particularly SPCs (which improve adherence), effective public health screening programs targeting patients to drive health-seeking behaviours, an increase in physician/patient awareness and early implementation of lifestyle changes. A unified Asia-Pacific guideline on hypertension management with pragmatic recommendations, particularly in resource-limited settings, is essential.

Trends in Medicare Reimbursement for Interventional Radiology Procedures: 2007-2020.

PURPOSE: Declining physician reimbursement has been occurring across multiple specialties due to changes in Medicare legislation, including the Deficit Reduction Omnibus Reconciliation Act (DRA), the Balanced Budget Act, and the Sustainable Growth Rate. The purpose of this study was to evaluate trends in Medicare reimbursement rates for various procedural classes in interventional radiology from 2007 to 2020. METHODS: Common interventional radiology procedures were selected across multiple procedural classes: gastrointestinal, biliary, urinary, fallopian dilatation, other injection/change/removal, iliac vascular, femoral/popliteal vascular, tibial/peroneal vascular, hepatobiliary, and vascular emergency. The Physician Fee Schedule Look-Up Tool from the Centers for Medicare & Medicaid Services was queried for current procedural terminology (CPT) codes to extract reimbursement data. All monetary data were adjusted for inflation using the United States consumer price index (CPI). The compound annual growth rate (CAGR) and average annual change in reimbursement were calculated based on these adjusted trends. RESULTS: Aside from urinary and vascular emergency procedures, all other procedural classes experienced decreases in inflation-adjusted Medicare reimbursement from 2007 to 2020. The greatest mean decrease in reimbursement rates was observed in biliary procedures (-$21.25), while the largest mean increase in reimbursement rates was observed in vascular emergency procedures ($3.23). All procedures with increases in reimbursement rates and 36.8% of procedures with decreases in reimbursement rates have a CPT code change between 2007 and 2020. CONCLUSION: After accounting for inflation, reimbursement rates were shown to decline for all procedural classes except for urinary and vascular emergencies. Congressional policies, such as the Deficit Reduction Act (DRA) and the Medicare Access and Children's Health Insurance Program (CHIP) Reauthorization Act of 2015, may clarify some of these trends.

Alignment, segmentation and neighborhood analysis in cyclic immunohistochemistry data using CASSATT.

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. Additionally, cycIHC benefits from the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the log odds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact-or do not interact-at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT is a fully open source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique.

Creation and validation of 3D-printed head molds for stereotaxic injections of neonatal mouse brains.

BACKGROUND: An increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory. NEW METHOD: A complete protocol and open-source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage. RESULTS: A series of test injections with adenovirus encoding red fluorescent protein, or Fluorogold, were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections. COMPARISON WITH EXISTING METHODS: The new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time. CONCLUSIONS: This 3D printing pipeline can be used to efficiently develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.

High-sensitivity troponin I for cardiovascular risk stratification in the general asymptomatic population: Perspectives from Asia-Pacific.

Cardiac troponin is a sensitive and specific biomarker for acute myocardial injury and has been used in the diagnosis of acute coronary syndromes, and has emerged as a tool for identifying high risk individuals for primary preventive therapy. Recent evidence has emerged indicating that high-sensitivity cardiac troponin assays, which allow robust detection of very low troponin concentrations, could detect subclinical injury in asymptomatic patients. On 24 March 2018, a group of cardiologists from the Asia Pacific region convened to review the data and discuss the potential utility of high-sensitivity troponin I (hsTnI) in the risk assessment of cardiovascular disease in the general population. The group recognized the immense burden of cardiovascular disease in the Asia-Pacific region, and the limitations of current risk stratification strategies. Data demonstrates that cardiac biomarkers like hsTnI could improve risk stratification, and thresholds for hsTnI in cardiovascular disease risk classification have been developed in Caucasian populations but not validated in Asian populations. There is an urgent need to improve cardiovascular risk assessment in the Asia Pacific general population, validate the Asian threshold of high risk and prove the utility of targeting these high-risk individuals for primary preventive strategies.

Comparative evaluation of the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin scaffold.

BACKGROUND: The present study aims to comparatively evaluate the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin (PRF) scaffold. MATERIALS AND METHODS: A total of 15 systemically healthy individuals between the age group of 15-25 years requiring third molar or orthodontic premolar extractions. Teeth were extracted atraumatically and transported to the laboratory. Stem cells were isolated from dental pulp and periodontal ligament. After attaining more than 90% confluency by the 7th day, these cells were tested for their viability and characterization. Stem cells were also incubated with PRF and viability was assessed on the 7th day. RESULTS: The mean number of cell for dental pulp stem cells (DPSCs) and periodontal ligament stem cell (PDLSC) was statistically insignificant (P > 0.05). The mean live cell viability was compared between DPSC (98.07%) and PDLSC (98%). Both DPSC and PDLSC showed a high percentage of expression of CD73 markers, 30.40% and 29.80%, respectively. However, DPSCs and PDLSCs lacked expression of CD34 expressing only 3.47% and 3.53%, respectively. PRF membrane as a scaffold exhibited no cytotoxic effects on DPCS's or PDLSC's. The cell viability of cells cultured with PRF was statistically insignificant (P > 0.05) when compared to the cells cultured with culture media. CONCLUSION: The study thus indicates that dental pulp and periodontal ligament are both rich sources of mesenchymal stem cells and can be successfully used for obtaining stem cells. PRF exhibits no cytotoxic effects on the cells and can be used in conjunction with dental stem cells.

Near fatal iron intoxication managed conservatively.

We report here the case of an infant who presented to the emergency department after unintentional ingestion of almost 130 mg/kg of elemental iron. The infant had evidence of serious toxicity in the form of hypotension, metabolic acidosis and excessive irritability. Her serum iron levels were 360 µg/dl, which was well above the normal range for her age. Despite such high serum levels, the infant made an uneventful recovery with medical management alone and did not require exchange transfusion. We chose to report this case to highlight her uneventful recovery with conservative therapy alone.

Frequency and predictors of contrast-induced nephropathy after angioplasty for chronic total occlusions.

BACKGROUND: The incidence and predictors of contrast-induced nephropathy (CIN) after percutaneous coronary intervention (PCI) of chronic total occlusions (CTO) have not been specifically reported. METHODS: This retrospective analysis included all consecutive patients referred for PCI of CTO between April 2003 and March 2008, with baseline and 24 h postprocedural available creatinine levels. CIN was defined as 24 h postprocedural increase of baseline creatinine levels > or =0.5 mg/dl (CIN(05)) or > or =25% (CIN(25)). Severe renal dysfunction (SRD) was defined as acute renal failure requiring dialysis, or an increase in baseline creatinine levels > or =2.0 mg/dl (SRD(2)) or > or =50% (SRD%). Patients were classified into risk categories for CIN, according to the validated Mehran risk score. RESULTS: A total of 227 patients were included, mean age of 64+/-10 years, the majority being at low risk for CIN (55% with < or =5 points in the Mehran score). CIN(25) occurred in 6.16% (14/227) patients and CIN(05) in 0.88% (2/227). The incidence of SRD(2) or SDR% was 0% (0/227) and 0.9% (2/227), respectively, with no patient requiring dialysis. Patients who developed CIN(25) received a higher contrast volume than those who did not (312 ml (210-400) vs 260 ml (200-345), p=0.14), but the difference was not statistically significant. CONCLUSIONS: In this consecutive cohort of patients, the incidence of CIN following PCI for CTO was low despite the administration of moderate to large volumes of contrast media. Attempts at revascularization of CTO should not be discouraged or be prematurely interrupted because of the fear of CIN.

Percutaneous coronary intervention and 30-day mortality: the British Columbia PCI risk score.

OBJECTIVES: To construct a calculator to assess the risk of 30-day mortality following PCI. BACKGROUND: Predictors of 30-day mortality are commonly used to aid management decisions for cardiac surgical patients. There is a need for an equivalent risk-score for 30-day mortality for percutaneous coronary intervention (PCI) as many patients are suitable for both procedures. METHODS: The British Columbia Cardiac Registry (BCCR) is a population-based registry that collects information on all PCI procedures performed in British Columbia (BC). We used data from the BCCR to identify risk factors for mortality in PCI patients and construct a calculator that predicts 30-day mortality. RESULTS: Patients (total n = 32,899) were divided into a training set (n = 26,350, PCI between 2000 and 2004) and validation set (n = 6,549, PCI in 2005). Univariate predictors of mortality were identified. Multivariable logistic regression analysis was performed on the training set to develop a statistical model for prediction of 30-day mortality. This model was tested in the validation set. Variables that were objective and available before PCI were included in the final risk score calculator. The 30-day mortality for the overall population was 1.5% (n = 500). Area under the ROC curve was 90.2% for the training set and 91.1% for the validation set indicating that the model also performed well in this group. CONCLUSIONS: We describe a large, contemporary cohort of patients undergoing PCI with complete follow-up for 30-day mortality. A robust, validated model of 30-day mortality after PCI was used to construct a risk calculator, the BC-PCI risk score, which can be accessed at www.bcpci.org.

Coronary steal with unstable angina secondary to a coronary artery fistula.

BACKGROUND: A 62-year old man with progressive deterioration of stable angina presented with an unstable episode and was referred for diagnostic coronary angiography. INVESTIGATIONS: Physical examination, electrocardiography, myocardial perfusion scan, coronary angiography, cardiac CT. DIAGNOSIS: Left coronary main stem to pulmonary artery fistula. MANAGEMENT: Coronary angiography, percutaneous coronary intervention, coil embolisation.

Tubercular myocarditis presenting with ventricular tachycardia.

BACKGROUND: A previously fit and healthy 30-year-old man reported experiencing palpitations accompanied by nausea, sweating and presyncope. These symptoms were found to be associated with episodes of nonsustained ventricular tachycardia. He was a nonsmoker, did not drink excessively, denied illicit drug use and had no family history of structural cardiac disease or sudden death. INVESTIGATIONS: Electrocardiography, laboratory tests, electrophysiological studies, echocardiography, coronary angiography, chest radiography, cardiac MRI (with late gadolinium enhancement), chest CT, lymph-node biopsy, Ziehl Nielsen staining, blood and sputum cultures and heaf testing. DIAGNOSIS: Tubercular myocarditis. MANAGEMENT: Antituberculous chemotherapy supported by antiarrhythmic and steroid pharmacotherapy and cardioverter-defibrillator implantation. Repeated imaging was performed to monitor disease progression.

Endothelial progenitor cells, late stent thrombosis and delayed re-endothelialisation.

A decade ago, the description of a primitive novel cell type capable of differentiating into cells expressing a mature endothelial cell -phenotype and their capacity to incorporate into regions of active angiogenesis, witnessed the emergence of endothelial progenitor cell (EPC) biology1. The development and maturation of this new concept in vascular biology has resulted in numerous studies describing the role of EPCs in a myriad of disease states where abnormalities of the vasculature have been implicated. Thus, from pre-eclampsia to pulmonary hypertension, erythropoietin administration to erectile dysfunction and cancer to coronary disease the discovery of EPCs has added greatly to the understanding of basic pathophysiology. However, it is in the study of coronary artery -disease where this paradigm shift has had greatest impact, not only regarding basic disease mechanisms, but in the rapid translation of these findings into a clinical context. The purpose of this review is to outline the current understanding of the EPC phenotypes and their relationship with risk factors for coronary disease. In addition, the potential problems of EPC dysfunction and its impact on percutaneous intervention will be appraised together with both pharmacological and stent based strategies to augment EPC -number and function.

Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1/Cip1-Cdk4-cyclin D1 assembly.

Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.

Vascular endothelial growth factor synergistically enhances induction of E-selectin by tumor necrosis factor-alpha.

OBJECTIVE: The regulation of endothelial cell adhesion molecules (CAMs) by vascular endothelial growth factor (VEGF) was investigated in cell cultures and in a rabbit model of atherogenic neointima formation. METHODS AND RESULTS: VEGF regulation of vascular CAM-1 (vascular cell adhesion molecule), intercellular CAM-1 (intercellular adhesion molecule), and E-selectin were investigated in human umbilical vein endothelial cells using quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry, and in the rabbit collar model of atherogenic macrophage accumulation by immunostaining. VEGF alone caused no significant induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, or E-selectin compared with tumor necrosis factor-alpha. In both hypercholesterolemic and normal rabbits, adenoviral VEGF-A165 expression caused no increase in endothelial vascular cell adhesion molecule-1 or E-selectin. In contrast, pretreatment of human umbilical vein endothelial cells with VEGF significantly increased E-selectin expression induced by tumor necrosis factor-alpha, compared with tumor necrosis factor-alpha alone, whereas vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 were unaffected. VEGF similarly enhanced IL-1beta-induced E-selectin upregulation. VEGF also synergistically increased tumor necrosis factor-alpha-induced E-selectin mRNA and shedding of soluble E-selectin. Synergistic upregulation of E-selectin expression by VEGF was mediated via VEGF receptor-2 and calcineurin signaling. CONCLUSIONS: VEGF alone does not activate endothelium to induce CAM expression; instead, VEGF "primes" endothelial cells, sensitizing them to cytokines leading to heightened selective pro-inflammatory responses, including upregulation of E-selectin.

Role of angiogenesis in cardiovascular disease: a critical appraisal.

The role of angiogenesis in atherosclerosis and other cardiovascular diseases has emerged as a major unresolved issue. Angiogenesis has attracted interest from opposite perspectives. Angiogenic cytokine therapy has been widely regarded as an attractive approach both for treating ischemic heart disease and for enhancing arterioprotective functions of the endothelium; conversely, a variety of studies suggest that neovascularization contributes to the growth of atherosclerotic lesions and is a key factor in plaque destabilization leading to rupture. Here, we critically review the evidence supporting a role for angiogenesis and angiogenic factors in atherosclerosis and neointima formation, emphasizing the problems raised by some of the landmark studies and the suitability of animal models of atherosclerosis and neointimal thickening for investigating the role of angiogenesis. Because many of the relevant studies have focused on the role of vascular endothelial growth factor (VEGF), we consider this work in the wider context of VEGF biology and in light of recent experience from clinical trials of VEGF and other angiogenic cytokines for ischemic heart disease. Also discussed are recent findings suggesting that, although angiogenesis may contribute to neointimal growth, it is not required for the initiation of intimal thickening. Our assessment of the evidence leads us to conclude that, although microvessels are a feature of advanced human atherosclerotic plaques, it remains unclear whether angiogenesis either plays a central role in the development of atherosclerosis or is responsible for plaque instability. Furthermore, current evidence from clinical trials of both proangiogenic and antiangiogenic therapies does not suggest that inhibition of angiogenesis is likely to be a viable therapeutic strategy for cardiovascular disease.

Placental growth factor promotes atherosclerotic intimal thickening and macrophage accumulation.

BACKGROUND: Placental growth factor (PlGF) has been implicated in the pathophysiological angiogenesis and monocyte recruitment that underlie chronic inflammatory disease, but its role in atherosclerosis has not been examined. We investigated the effects of exogenous PlGF, delivered by adenoviral gene transfer, on atherogenic intimal thickening and macrophage accumulation induced by collar placement around the rabbit carotid artery and examined the effects of PlGF deficiency on atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Periadventitial transfer of PlGF2-encoding adenoviruses significantly increased intimal thickening, macrophage accumulation, endothelial vascular cell adhesion molecule-1 expression, and adventitial neovascularization in the collared arteries of hypercholesterolemic rabbits and increased the intima-to-media ratio in rabbits fed a normal diet. Neointimal macrophages were associated with increased expression of the PlGF receptor Flt-1. The size and macrophage content of early atherosclerotic lesions were reduced in mice deficient in both apoE and PlGF compared with apoE-deficient mice. CONCLUSIONS: Local adenoviral PlGF2 delivery promotes atherogenic neointima formation in hypercholesterolemic rabbits, and PlGF is required for macrophage infiltration in early atherosclerotic lesions in apoE(-/-) mice. These findings support a novel role for PlGF in the pathogenesis of atherosclerotic disease.

Angiogenesis-dependent and independent phases of intimal hyperplasia.

BACKGROUND: Neointimal vascular smooth muscle cell (VSMC) proliferation is a primary cause of occlusive vascular disease, including atherosclerosis, restenosis after percutaneous interventions, and bypass graft stenosis. Angiogenesis is implicated in the progression of early atheromatous lesions in animal models, but its role in neointimal VSMC proliferation is undefined. Because percutaneous coronary interventions result in induction of periadventitial angiogenesis, we analyzed the role of this process in neointima formation. METHODS AND RESULTS: Local injury to the arterial wall in 2 different animal models induced periadventitial angiogenesis and neointima formation. Application of angiogenesis stimulators vascular endothelial growth factor (VEGF-A165) or a proline/arginine-rich peptide (PR39) to the adventitia of the injured artery induced a marked increase in neointimal thickening beyond that seen with injury alone in both in vivo models. Inhibition of either VEGF (with soluble VEGF receptor 1 [sFlt1]) or fibroblast growth factor (FGF) (with a dominant=negative form of FGF receptor 1 [FGF-R1DN]), respectively, signaling reduced adventitial thickening induced by VEGF and PR39 to the level seen with mechanical arterial injury alone. However, neither inhibitor was effective in preventing neointimal thickening after mechanical injury when administered in the absence of angiogenic growth factor. CONCLUSIONS: Our findings indicate that adventitial angiogenesis stimulates intimal thickening but does not initiate it.