Artery Specimens Research Articles

Bidirectional roles of extracellular and intracellular HMGB2 in the development of atherosclerosis

Abstract Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized. Objective The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro. Methods/Results We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice. Conclusion This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.

A-342 Precision Evaluation of the pH, PO2 and PCO2 tests in the i-STAT® G3+ Cartridge using the i-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings

Abstract Background Precise and accurate measurements of blood gases are important for the diagnosis, monitoring and treatment of respiratory, metabolic, and acid-base disturbances. This study demonstrates the precision of the pH, partial pressure of oxygen (PO2), and partial pressure of carbon dioxide (PCO2) tests in the i-STAT G3+ cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point-of-care or clinical laboratory settings. Methods The precision of the i-STAT pH, PO2, and PCO2 tests in the i-STAT G3+ cartridge was assessed at external clinical sites by end-users using whole blood specimens across the measurement range for each test: pH (6.500 - 7.800 pH units), PO2 (5 - 700 mmHg), PCO2 (5.0 - 130.0 mmHg). The study included venous whole blood samples from ∼140 adult subjects (≥ 18 years) and arterial whole blood samples from ∼140 adult subjects at 3 clinical sites. Each whole blood sample was run in duplicate on the i-STAT G3+ cartridge. In addition, ∼140 specimens were collected in capillary tubes using two fingerstick draws from adult subjects and ∼20 capillary heel-stick specimens were collected into two capillary tubes from neonate subjects (birth - 28 days) at two clinical sites. Specimens used in this study were prospectively collected venous, arterial, or capillary specimens under an Institutional Review Board approved informed consent or were de-identified leftover venous or arterial specimens. For each specimen type, duplicates from each subject were grouped into subintervals based on their mean values. The subintervals included the medical decision levels of 7.300, 7.350 and 7.450 pH units for pH, 30, 45 and 60 mmHg for PO2 and 35, 45, 50 and 70 mmHg for PCO2. The estimates of the pooled standard deviation (SD) and pooled coefficient of variation (%CV), as well as their respective 95% confidence intervals were calculated for each subinterval. Results Across all specimen types (excluding outliers), the pooled SD for pH ranged from 0.00591 - 0.01747 pH units for subintervals 6.500 - 7.300, >7.300 - 7.450, >7.450 - 7.800 pH units. For PO2, the pooled SD ranged from 0.71 - 3.76 mmHg for subintervals 10 - 40 mmHg and >40 - 50 mmHg and the pooled %CV ranged from 1.63 - 8.76 %CV for subintervals >50 - 100 mmHg, >100 - 250 mmHg and >250 - 700 mmHg. For PCO2, the pooled SD ranged from 0.000 - 1.709 mmHg for subintervals 5.0 - 35.0 mmHg, >35.0 - 50.0 mmHg and >50.0 - 62.5 mmHg and the pooled %CV ranged from 1.10 - 3.62 %CV for subinterval >62.5 - 130.0 mmHg. Conclusions The precision study demonstrated that the pH, PO2 and PCO2 tests in the i-STAT G3+ cartridge when tested using the i-STAT 1 System deliver precise results within ∼2 minutes for venous, arterial, and capillary whole blood patient specimens when tested by end users in a point-of-care or clinical laboratory setting. These studies were funded by Abbott Laboratories.

A-328 Accuracy of the Glucose Test in the i-STAT® G Cartridge Using the i-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings

Abstract Background Precise and accurate measurements of whole blood glucose levels are important for the diagnosis, monitoring and treatment of carbohydrate metabolism disorders, such as diabetes and hypoglycemia. This study demonstrates the accuracy of the glucose test in the i-STAT G cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point of care or clinical laboratory settings. Methods The accuracy of the glucose test in the i-STAT G cartridge was assessed at external clinical sites in a method comparison study using whole blood specimens across the measurement range of 20 - 700 mg/dL. Specimens used in this study were venous, arterial, and capillary specimens prospectively collected under Institutional Review Board-approved informed consent or were leftover de-identified venous or arterial specimens. The study included venous whole blood samples from 164 adult subjects (≥ 18 years) and arterial whole blood samples from 173 adult subjects at 3 clinical sites, and capillary whole blood samples from 211 adult subject at 3 clinicals sites and 23 neonate subjects (birth to 28 days) at 2 clinical sites. For venous and arterial whole blood, samples were collected and tested in duplicate on the i-STAT G cartridge and on the i-STAT CHEM8+ cartridge (comparator). For capillary whole blood samples, adult fingerstick samples and neonate heel-stick samples were collected in capillary tubes and tested in singlicate on the i-STAT G cartridge and in singlicate on the epoc® Blood Analysis System (comparator). Passing-Bablok linear regression was performed for venous and arterial whole blood samples using the first replicate from the i-STAT G cartridge versus the mean result of the comparator, and for capillary whole blood samples (adult and neonate combined) using the singlicate result from the i-STAT G cartridge versus the singlicate result of the comparator. Results Passing-Bablok regression of i-STAT G cartridge results versus the comparator was used to calculate slope and correlation coefficient (r). Adult venous whole blood results had slope = 1.00 and r = 1.00, and adult arterial whole blood results had slope = 1.00 and r = 1.00. Adult and neonate capillary whole blood results had slope = 1.00 and r = 1.00. Conclusions These results demonstrate that the i-STAT 1 System delivers accurate glucose test results within ∼2 minutes using the i-STAT G cartridge for venous, arterial and capillary whole blood samples for adult and neonate subjects across a glucose range of 20-700 mg/dL when tested by end-users in a point of care or clinical laboratory setting. This study was funded by Abbott Laboratories.

A-329 Accuracy of the pH, PO2, and PCO2 Tests in the i-STAT® G3+ Cartridge Using the i-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings

Abstract Background Precise and accurate measurements of blood gases are important for the diagnosis, monitoring and treatment of respiratory, metabolic, and acid-base disturbances. This study demonstrates the accuracy of the pH, partial pressure of oxygen (PO2), and partial pressure of carbon dioxide (PCO2) tests in the i-STAT G3+ cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point of care or clinical laboratory settings. Methods The accuracy of the PO2, and PCO2 tests in the i-STAT G3+ cartridge was assessed at external clinical sites in a method comparison study using whole blood specimens across the measurement range for each test: pH 6.5-7.8, PO2 5-700 mmHg, PCO2 5.0-130.0 mmHg. Specimens used in this study were venous, arterial, and capillary specimens prospectively collected under Institutional Review Board-approved informed consent or were leftover de-identified venous or arterial specimens. The study included venous and arterial whole blood samples from ∼140 adult subjects (≥ 18 years) at 3 clinical sites, and capillary whole blood samples from ∼180 adult subject at 3 clinicals sites and ∼25 neonate subjects (birth to 28 days) at 2 clinical sites. The RapidPoint System was used as comparator. For venous and arterial whole blood, samples were collected and tested in duplicate on the i-STAT G3+ cartridge and in singlicate on the RapidPoint System. For capillary whole blood samples, adult fingerstick samples and neonate heel-stick samples were collected in capillary tubes and tested in singlicate on the i-STAT G3+ cartridge and on the RapidPoint System. Passing-Bablok regression was performed for venous and arterial whole blood samples combined, using the first replicate from the i-STAT G3+ cartridge versus the singlicate result from the comparator, and for capillary whole blood samples (adult and neonate combined) using the singlicate result from the i-STAT G3+ cartridge versus the singlicate result from the comparator. Results Passing-Bablok regression of i-STAT G3+ cartridge results versus the comparator was used to calculate slope and correlation coefficient (r). For adult venous and arterial whole blood results, pH had slope = 0.96 and r = 0.99, PO2 had slope = 1.05 and r = 1.00, and PCO2 had slope = 1.04 and r = 0.98. For adult and neonate capillary whole blood results, pH had slope = 1.02 and r = 0.98, PO2 had slope = 1.09 and r = 0.99, and PCO2 had slope = 1.07 and r = 0.96. Conclusions These results demonstrate that the i-STAT 1 System delivers accurate pH, PO2, and PCO2 test results with ∼2 minutes using the i-STAT G3+ cartridge for venous, arterial and capillary whole blood samples for adult and neonate subjects when tested by end-users in a point of care or clinical laboratory setting. This study was funded by Abbott Laboratories.

A-341 Precision Evaluation of the i-STAT® Glucose Test in the i-STAT G Cartridge using thei-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings

Abstract Background Precise and accurate measurements of whole blood glucose levels are important for the diagnosis and treatment of patients suffering from diabetes and hypoglycemia. This study demonstrates the precision of the i-STAT G cartridge with the i-STAT 1 System in arterial, venous, or capillary whole blood in point of care or clinical laboratory settings. Methods The precision of the i-STAT G cartridge was assessed at external clinical sites by intended end users with whole blood specimens across the measurement range of 20 - 700 mg/dL. The study included testing venous whole blood samples from 154 adult (≥ 18 years) patients and arterial whole blood samples from 173 adult patients at three clinical sites. Each whole blood sample was run in duplicate on the i-STAT G cartridge. In addition, 139 capillary whole blood specimens from both adult subjects and neonate subjects (birth - 28 days) were collected and tested for capillary whole blood precision. Duplicate fingerstick draws were collected in capillary tubes from 119 adult subjects at two clinical sites and duplicate heel-stick specimens were collected in capillary tubes from 20 neonate subjects at two clinical sites. Specimens used in this study were prospectively collected venous, arterial, or capillary specimens under an Institutional Review Board approved informed consent or were de-identified leftover venous or arterial specimens. For each specimen type, duplicates from each subject were grouped into subintervals based on their mean values. The subintervals included the glucose medical decision levels of 45 mg/dL, 120 mg/dL and 180 mg/dL. The estimates of the pooled standard deviation (SD) and pooled coefficient of variation (%CV), as well as their respective 95% confidence intervals (CI) were calculated for each subinterval (20-90 mg/dL, >90-150 mg/dL, >150-250 mg/dL, >250-700 mg/dL or >250-400 mg/dL and >400-700 mg/dL, as applicable). The precision estimates for each subinterval were compared against an acceptance criteria of the greater of 3 mg/dL or 5% of the mean (mg/dL) of the subinterval. Results The whole blood precision was found to be less than or equal to 5.00 %CV for all subintervals for each specimen type. For venous specimens, the %CV ranged from 0.35% to 0.43%; for arterial specimens the %CV ranged from 0.26% to 0.46% and for capillary specimens (adult and neonate combined), the %CV ranged from 1.38% to 2.71%. Conclusions The study demonstrated that the i-STAT Glucose test in the i-STAT G cartridge when tested using the i-STAT 1 System delivers precise results within ∼2 minutes for venous, arterial, and capillary whole blood patient specimens across a glucose range of 20 - 700 mg/dL when tested by end users in a point of care or clinical laboratory setting. The studies were funded by Abbott Laboratories.

A-333 Comparison between measured total carbon dioxide and calculated bicarbonate in hospitalized patients

Abstract Background Bicarbonate (HCO3-) is a key indicator of anion excess or deficit and important clinically for assessing acid-base balance. For patient care, HCO3- may be reported as a calculated value from blood gas analysis or measured on automated chemistry analyzers. When measured, the final result includes HCO3-, carbonate (CO32-) and carbamino compounds and thus is reported as total CO2 (TCO2). Anecdotally, some patients have discrepancies between HCO3- and TCO2. However, there is limited data in the published literature assessing agreement between the two methods. Our study aims to compare the calculated HCO3- values with measured TCO2 obtained from simultaneously collected clinical specimens. Methods This study included all results from physician ordered HCO3- and TCO2 between 01/2019 to 12/2020 retrospectively retrieved from the laboratory information system (Cerner). Specimens were collected by nursing staff within 5 minutes of each other. TCO2 was assessed on a Roche cobas 702 (CV = 4.2%) according to the manufacturer’s instructions using an enzymatic phosphoenolpyruvate carboxylase method in lithium heparin tubes. HCO3- was calculated using a Radiometer ABL800 blood gas analyzer from arterial or venous blood gas samples according to the manufacturer’s instructions. Briefly, HCO3- is calculated using the Henderson-hasselbalch equation and the measurement of PCO2 (CV= 1.75%), and pH (CV = 0.06%). Deming regression was performed. The CLIA total allowable error of 4 mmol/L and a reference change value (RCV, Cv Individual = 4%) of -13 and + 14 for TCO2 was used to assess agreement. All data was analyzed in R. Results There were 101,895 paired HCO3- and TCO2 results during the study period. The values ranged from 4 to 56 mmol/L and the mean was 25.7. The mean difference between the calculated HCO3- and measured TCO2 was 1.33 mmol/L. Relative to HCO3-, there were 2,119 (2.1%) TCO2 results with a negative bias exceeding 4 mmol/L and 427 (0.4%)[FC1] with a positive bias exceeding 4 mmol/L. Twenty-two samples exceeded the positive RCV of 14% and 22 samples exceeded the negative RCV. Deming regression revealed a slope of 1.09 (95% CI: 1.087-1.092) an intercept of -2.19 (-2.18 to -2.06), and a Pearson correlation of 0.960 (0.959 to 0.960). We also analyzed differences between HCO3- and TCO2 in arterial and venous samples. The Pearson r for venous samples was 0.956 (0.955 to 0.957) and for arterial samples was 0.966 (0.966-0.966). Deming regression revealed a slope of 1.099 (0.195 to 1.104) and 1.071 (1.068 to 1.074) for venous and arterial samples respectively. The intercept for venous samples was -1.441 (-1.559 to -1.324) and for arterial was -2.005 (-2.072 to -1.938). Conclusions There is bias between HCO3- calculations and TCO2 measurements in simultaneously drawn samples. A small proportion of samples (2.5%) exceed the CLIA total allowable error between the results but very few exceed the RCV. This may due to differences in matrices, with different correlations and slopes observed between arterial and venous specimens. Further studies are required to distinguish the causes of the observed differences between HCO3- and TCO2.

Ubiquitination of angiotensin-converting enzyme 2 contributes to the development of pulmonary arterial hypertension mediated by neural precursor cell–expressed developmentally down-regulated gene 4-Like

ObjectivesIn this study, we investigated whether neural precursor cell–expressed developmentally down-regulated gene 4-like (NEDD4L) is the E3 enzyme of angiotensin-converting enzyme 2 (ACE2) and whether NEDD4L degrades ACE2 via ubiquitination, leading to the progression of pulmonary arterial hypertension (PAH).MethodsBioinformatic analyses were used to explore the E3 ligase that ubiquitinates ACE2. Cultured pulmonary arterial smooth muscle cells (PASMCs) and specimens from patients with PAH were used to investigate the crosstalk between NEDD4L and ACE2 and its ubiquitination in the context of PAH.ResultsThe inhibition of ubiquitination attenuated hypoxia-induced proliferation of PASMCs. The levels of NEDD4L were increased, and those of ACE2 were decreased in lung tissues from patients with PAH and in PASMCs. NEDD4L, the E3 ligase of ACE2, inhibited the expression of ACE2 in PASMCs, possibly through ubiquitination-mediated degradation. PAH was associated with upregulation of NEDD4L expression and downregulation of ACE2 expression.ConclusionsNEDD4L, the E3 ubiquitination enzyme of ACE2, promotes the proliferation of PASMCs, ultimately leading to PAH.

Dissecting contributions of pulmonary arterial remodeling to right ventricular afterload in pulmonary hypertension.

Pulmonary hypertension (PH) is defined as an elevation in the right ventricle (RV) afterload, characterized by increased hemodynamic pressure in the main pulmonary artery (PA). Elevations in RV afterload increase RV wall stress, resulting in RV remodeling and potentially RV failure. From a biomechanical standpoint, the primary drivers for RV afterload elevations include increases in pulmonary vascular resistance (PVR) in the distal vasculature and decreases in vessel compliance in the proximal PA. However, the individual contributions of the various vascular remodeling events toward the progression of PA pressure elevations and altered vascular hemodynamics remain elusive. In this study, we used a subject-specific one-dimensional (1D) fluid-structure interaction (FSI) model to investigate the alteration of pulmonary hemodynamics in PH and to quantify the contributions of vascular stiffening and increased resistance towards increased main pulmonary artery (MPA) pressure. We used a combination of subject-specific hemodynamic measurements, ex-vivo mechanical testing of arterial tissue specimens, and ex-vivo X-ray micro-tomography imaging to develop the 1D-FSI model and dissect the contribution of PA remodeling events towards alterations in the MPA pressure waveform. Both the amplitude and pulsatility of the MPA pressure waveform were analyzed. Our results indicated that increased distal resistance has the greatest effect on the increase in maximum MPA pressure, while increased stiffness caused significant elevations in the characteristic impedance. The method presented in this study will serve as an essential step toward understanding the complex interplay between PA remodeling events that leads to the most severe adverse effect on RV dysfunction.

The role of 6-phosphogluconate dehydrogenase in vascular smooth muscle cell phenotypic switching and angioplasty-induced intimal hyperplasia

BackgroundRestenosis poses a significant challenge for individuals afflicted with peripheral artery diseases, often leading to considerable morbidity and necessitating repeated interventions. The primary culprit behind the pathogenesis of restenosis is intimal hyperplasia (IH), in which the hyperproliferative and migratory vascular smooth muscle cell (VSMC) accumulate excessively in the tunica intima. 6-Phosphogluconate dehydrogenase (6PGD), sometimes referred to as PGD, is one of the critical enzymes in pentose phosphate pathway (PPP). In this study, we sought to probe whether 6PGD is aberrantly regulated in IH and contributes to VSMC phenotypic switching. MethodsWe used clinical specimens of diseased human coronary arteries with IH lesions and observed robust upregulation of 6PGD at protein level in both the medial and intimal layers in comparison with healthy arterial segments. Results6PGD activity and protein expression were profoundly stimulated upon platelet-derived growth factor-induced VSMC phenotypic switching. Using gain-of-function (dCas9-mediated transcriptional activation) and loss-of-function (small interfering RNA-mediated) silencing, we were able to demonstrate the pathogenic role of 6PGD in driving VSMC hyperproliferation, migration, dedifferentiation, and inflammation. Finally, we conducted a rat model of balloon angioplasty in the common carotid artery, with Pluronic hydrogel-assisted perivascular delivery of Physcion, a selective 6PGD inhibitor with poor systemic bioavailability, and observed effective mitigation of IH. ConclusionsWe contend that aberrant 6PGD expression and activity—indicative of a metabolic shift toward pentose phosphate pathway—could serve as a new disease-driving mechanism and, hence, an actionable target for the development of effective new therapies for IH and restenosis after endovascular interventions.

Cadaveric Study of Colon-Lengthening Maneuvers After Sigmoidectomy.

Creation of a tension-free colorectal anastomosis after left colon resection or low anterior resection is a key requirement for technical success. The relative contribution of each of a series of known lengthening maneuvers remains incompletely characterized. The aim of this study was to compare technical procedures for lengthening of the left colon before rectal anastomosis. A series of lengthening maneuvers was performed on 15 fresh cadavers. Mean distance gained was measured for each successive maneuver, including 1) high inferior mesenteric artery ligation, 2) splenic flexure takedown, and 3) high inferior mesenteric vein ligation by the ligament of Treitz. Cadaveric study. The premobilization and postmobilization position of the proximal colonic end was measured relative to the inferior edge of the sacral promontory. Measurements of the colonic length relative to the sacral promontory were taken after each mobilization maneuver. The inferior mesenteric artery, sigmoid colon, and rectum specimen lengths were measured. The distance from the inferior border of the sacral promontory to the pelvic floor was measured along the sacral curvature. Mean sigmoid colon resection length was 34.7 ± 11.1 cm. Before any lengthening, baseline reach was -1.3 ± 4.2 cm from the sacral promontory. Inferior mesenteric artery ligation yielded an additional 11.5 ± 4.7 cm. Subsequent splenic flexure takedown added an additional 12.8 ± 9.6 cm. Finally, inferior mesenteric vein ligation added an additional 11.33 ± 6.9 cm, bringing the total colonic length to 35.7 ± 14.7 cm. BMI and weight negatively correlated with length gained. The study was limited by nature of being a cadaver study. Stepwise lengthening maneuvers allow significant additional reach to allow a tension-free left colon to rectal anastomosis. See Video Abstract . ANTECEDENTES:La creación de una anastomosis colorrectal libre de tensión tras una resección de colon izquierdo o tras una resección anterior baja es un requisito clave para el éxito relacionado con la técnica quirúrgica. La relativa contribución de las diversas maniobras de alargamiento permanece caracterizada de manera incompleta.OBJETIVO:El propósito de este estudio fue la de comparar procedimientos técnicos de alargamiento del colon izquierdo previo a la anastomosis rectal.DISEÑO:Una serie de maniobras de alargamiento fueron realizados en 15 cadáveres frescos. La distancia promedio ganada fue medida para cada maniobra sucesiva, incluyendo (1) ligadura alta de la arteria mesentérica inferior, (2) descenso del ángulo esplénico, (3) ligadura alta de la vena mesentérica interior mediante el ligamento de Treitz.AJUSTES:Estudio cadavérico.PRINCIPALES MEDIDAS DE RESULTADO:La posición premobilizacion y postmobilizacion del extremo proximal del colon fue medido tomando en cuenta el borde inferior del promontorio sacro. Las mediciones de la longitud colónica en relación al sacro fueron tomadas luego de cada maniobra de movilización. Fueron tomadas así mismo las longitudes de la arteria mesentérica inferior, el colon sigmoides y recto. Las distancias desde el borde inferior del promontorio sacro al suelo pelvico fueron medidas a lo largo de la curvatura sacra.RESULTADOS:Average sigmoid colon resection length was 34.7 ± 11.1 cm. Prior to any lengthening, baseline reach was -1.3 ± 4.2 cm from the sacral promontory. Inferior mesenteric artery ligation yielded an additional 11.5 ± 4.7 cm. Subsequent splenic flexure takedown added an additional 12.8 ± 9.6 cm. Finally, inferior mesenteric vein ligation added an additional 11.33 ± 6.9 cm, bringing the total colonic length to 35.7 ± 14.7 cm. BMI and weight negatively correlated with length gained.LIMITACIONES:Este estudio tuvo como limitación la naturaleza de haber sido un estudio cadavérico.CONCLUSIONES:Maniobras de alargamiento permiten un alcance adicional significativo permitiendo de esta manera una anastomosis de colon izquierdo a recto libre de anastomosis. (Traducción-Dr Osvaldo Gauto ).

Angiopoietin-2 associates with poor prognosis in Moyamoya angiopathy.

Moyamoya angiopathy (MA) is a rare cerebrovascular disorder characterized by recurrent ischemic/hemorrhagic strokes due to progressive occlusion of the intracranial carotid arteries. The lack of reliable disease severity biomarkers led us to investigate molecular features of a Caucasian cohort of MA patients. The participants consisted of 30 MA patients and 40 controls. We measured cerebrospinal fluid (CSF) levels of angiogenic/inflammatory factors (ELISA). We then applied quantitative real-time PCR on cerebral artery specimens for expression analyses of angiogenic factors. By an immunoassay based on microfluidic technology, we examined the potential correlations between plasma protein expression and MA clinical progression. A RNA interference approach toward Ring Finger Protein 213 (RNF213) and a tube formation assay were applied in cellular model. We detected a statistically significant (p < 0.000001) up-regulation of Angiopoietin-2 (Ang-2) in CSF and stenotic middle cerebral arteries (RQ >2) of MA patients compared to controls. A high Ang-2 plasma concentration (p = 0.018) was associated with unfavorable outcome in a subset of MA patients. ROC curve analyses indicated Ang-2 as diagnostic CSF biomarker (>3741 pg/mL) and prognostic plasma biomarker (>1162 pg/mL), to distinguish stable-from-progressive MA. Consistently, MA cellular model showed a significant up-regulation (RQ >2) of Ang-2 in RNF213 silenced condition. Our results pointed out Ang-2 as a reliable biomarker mirroring arterial steno-occlusion and vascular instability of MA in CSF and blood, providing a candidate factor for patient stratification. This pilot study may pave the way to the validation of a biomarker to identify progressive MA patients deserving a specific treatment path.

Single-cell ‘omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Single-cell 'omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations.

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-β (transforming growth factor-β) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-β family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-βR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-β-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-β signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-β signaling by downregulating TGF-β expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-β signaling in PASMCs. Such rebalance of BMP/TGF-β pathways is translationally important for PAH alleviation.

Mechanical, structural, and morphological differences in the iliac arteries

Iliac arteries play a crucial role in peripheral blood circulation. They are susceptible to various diseases, including aneurysms and atherosclerosis. Structure, material properties, and biomechanical forces acting on different regions of the iliac vasculature may contribute to the localization and progression of these pathologies. We examined 33 arterial specimens from common iliac (CI), external iliac (EI), and internal iliac (II) arteries obtained from 11 human donors (62 ± 12 years). We conducted morphometric, mechanical, and structural analyses using planar biaxial tests, constitutive modeling, and bi-directional histology on transverse and axial sections. The iliac arteries exhibited increased tortuosity and varying disease distribution with age. CI and II arteries displayed non-uniform age-related disease progression around their circumference, while EI remained healthy even in older individuals. Trends in load-free and stress-free thickness varied along the iliac vasculature. Longitudinally, EI exhibited the highest compliance compared to other iliac vessels. In contrast, CI was stiffest longitudinally, and EI was the stiffest circumferentially. Material parameters for all iliac vessels are reported for four common constitutive relations. Elastin near the internal elastic lamina displayed greater waviness in EI and II compared to CI. Also, EI had the least glycosaminoglycans (GAGs) and the highest elastin content. Our findings highlight variations in the morphological, mechanical, and structural properties of iliac arteries along their length. This data can inform vascular disease development and computational studies, and guide the development of biomimetic repair materials and devices tailored to specific iliac locations, improving vascular repair strategies.

Pathology of Critical Limb Ischemia; Comparison of Plaque Characteristics Between Anterior and Posterior Tibial Arteries.

Though the number of patients with peripheral arterial disease (PAD) and critical limb ischemia (CLI) is increasing, few histopathological studies of PAD, particularly that involving below-the-knee arteries, has been reported. We analyzed the pathology of anterior tibial artery (ATA) and posterior tibial artery (PTA) specimens obtained from patients who underwent lower extremity amputation due to CLIMethods: Dissected ATAs and PTAs were subjected to ex-vivo soft X-ray radiography, followed by pathological examination using 860 histological sections. This protocol was approved by the Ethics Review Board of Nihon University Itabashi Hospital (RK-190910-01) and Kyorin University Hospital (R02-179). The calcified area distribution was significantly larger in PTAs than in ATAs on soft X-ray radiographic images (ATAs, 48.3% ±19.2 versus PTAs, 61.6% ±23.9; p＜0.001). Eccentric plaque with necrotic core and macrophage infiltration were more prominent in ATAs than in PTAs (eccentric plaque: ATAs, 63.7% versus PTAs, 49.1%; p＜0.0001, macrophage: ATAs, 0.29% [0.095 - 1.1%] versus PTAs, 0.12% [0.029 - 0.36%]; p＜0.001), histopathologically. Thromboembolic lesions were more frequently identified in PTAs than in ATAs (ATAs, 11.1% versus PTAs 15.8%; p＜0.05). Moreover, post-balloon injury pathology differed between ATAs and PTAs. Histological features differed strikingly between ATAs and PTAs obtained from CLI patients. Clarifying the pathological features of CLI would contribute to establishing therapeutic strategies for PAD, particularly disease involving below-the knee-arteries.

Assessment of plasma soluble Tie-2 level to distinguish moyamoya disease from atherosclerotic cerebrovascular disease and predict postoperative neovascularization.

Moyamoya disease (MMD) is a chronic steno-occlusive cerebrovascular disease and features the formation of hazy collateral vessels at the base of the brain. Angiopoietin (Ang)-1 and -2, their receptor Tie-2, and vascular endothelial growth factor (VEGF) that regulate angiogenesis might be important in MMD pathophysiology and postoperative collateral formation. The goal of this study was to determine whether levels of these angiogenic factors could predict collateralization in patients with MMD. A total of 196 patients with MMD and 57 with atherosclerotic cerebrovascular disease serving as controls were enrolled. Ang-1, Ang-2, Tie-2, and VEGF mRNA levels were analyzed in middle cerebral artery (MCA) arterial wall specimens by using real-time quantitative polymerase chain reaction. MCA and peripheral plasma concentrations of Ang-1, Ang-2, soluble Tie-2 (sTie-2), and VEGF were examined by enzyme-linked immunosorbent assay. Cerebral arteriography was performed 6 months after bypass surgery to assess the postoperative collateralization. In MCA specimens, patients with MMD exhibited higher expression levels of Ang-1 and Ang-2 but lowered VEGF expression. The patients with MMD had significantly higher concentrations of Ang-1 and Ang-2 but lower levels of VEGF in MCA plasma. Peripheral plasma concentrations of these growth factors were not changed. MCA and peripheral plasma sTie-2 levels were both reduced in patients with MMD. The 6-month follow-up showed that patients with good collateral formation had lower sTie-2 levels in both MCA and peripheral plasma. Furthermore, the Suzuki stage and peripheral plasma sTie-2 level were significantly correlated with good postoperative collateral formation on multivariate analysis. Ang-1, Ang-2, Tie-2, and VEGF are involved in MMD pathogenesis. The peripheral plasma level of sTie-2 can differentiate MMD from atherosclerotic cerebrovascular disease and serve as a novel biomarker to predict postoperative collateral formation.

Thrombus Aspirates From Patients With Acute Ischemic Stroke Are Infiltrated by Viridans Streptococci.

Acute ischemic stroke may be due to embolism from ruptured atherosclerotic carotid arteries. DNA of oral bacteria, mainly the viridans streptococci group, has been detected in thrombus aspirates of patients with ischemic stroke as well as in carotid endarterectomy samples. Because viridans streptococci are known to possess thrombogenic properties, we studied whether their presence in thrombus aspirates and in carotid artery specimens can be confirmed using bacterial immunohistochemistry. Thrombus aspirates from 61 patients with ischemic stroke (70.5% men; mean age, 66.8 years) treated with mechanical thrombectomy, as well as carotid endarterectomy samples from 20 symptomatic patients (65.0% men; mean age, 66.2 years) and 48 carotid artery samples from nonstroke autopsy cases (62.5% men; mean age, 66.4 years), were immunostained with an antibody cocktail against 3 species (Streptococcus sanguinis, Streptococcus mitis, and Streptococcus gordonii) of viridans streptococci. Of the thrombus aspirates, 84.8% were immunopositive for viridans streptococci group bacteria, as were 80.0% of the carotid endarterectomy samples, whereas immunopositivity was observed in 31.3% of the carotid artery samples from nonstroke autopsies. Most streptococci were detected inside neutrophil granulocytes, but there were also remnants of bacterial biofilm as well as free bacterial infiltrates in some samples. Oral streptococci were found in aspirated thrombi of patients with acute ischemic stroke as well as in carotid artery samples. Our results suggest that viridans streptococci group bacteria may play a role in the pathophysiology of ischemic stroke.

Upregulated Cytoskeletal Proteins Promote Pathological Angiogenesis in Moyamoya Disease.

Moyamoya disease (MMD) is a rare progressive vascular disease that leads to intracranial internal carotid artery stenosis and eventual occlusion. However, its pathogenesis remains unclear. The purpose of this study is to explore the role of abnormally expressed proteins in the pathogenesis of MMD. Data-independent acquisition mass spectrometry identifies the differentially expressed proteins in MMD serum by detecting the serum from 60 patients with MMD and 20 health controls. The differentially expressed proteins were validated using enzyme linked immunosorbent assays. Immunofluorescence for superficial temporal artery and middle cerebral artery specimens was used to explore the morphological changes of vascular wall in MMD. In vitro experiments were used to explore the changes and mechanisms of differentially expressed proteins on endothelial cells. Proteomic analysis showed that a total of 14 726 peptides and 1555 proteins were quantified by mass spectrometry data. FLNA (filamin A) and ZYX (zyxin) proteins were significantly higher in MMD serum compared with those in health controls (Log2FC >2.9 and >2.8, respectively). Immunofluorescence revealed an intimal hyperplasia in superficial temporal artery and middle cerebral artery specimens of MMD. FLNA and ZYX proteins increased the proportion of endothelial cells in S phase and promoted their proliferation, angiogenesis, and cytoskeleton enlargement. Mechanistic studies revealed that AKT (serine/threonine kinase)/GSK-3β (glycogen synthase kinase 3β)/β-catenin signaling pathway plays a major role in these FLNA- and ZYX-induced changes in endothelial cells. This study provides proteomic data on a large sample size of MMD. The differential expression of FLNA and ZYX in patient with MMD and following in vitro experiments suggest that these upregulated proteins are related to the pathology of cerebrovascular intimal hyperplasia in MMD and are involved in MMD pathogenesis, with diagnostic and therapeutic ramifications.

Ultrasound-guided periadventitial administration of rapamycin-fibrin glue attenuates neointimal hyperplasia in the rat carotid artery injury model

IntroductionArterial restenosis caused by intimal hyperplasia (IH) is a serious complication after vascular interventions. In the rat carotid balloon injury model, we injected phosphate buffer saline (PBS), rapamycin-phosphate buffer saline suspension (RPM-PBS), blank fibrin glue (FG) and rapamycin-fibrin glue (RPM-FG) around the injured carotid artery under ultrasound guidance and observed the inhibitory effect on IH. MethodsThe properties of RPM-FG in vitro were verified by scanning electron microscopy (SEM) and determination of the drug release rate. FG metabolism in vivo was observed by fluorescence imaging. The rat carotid balloon injury models were randomly classified into 4 groups: PBS group (control group), RPM-PBS group, FG group, and RPM-FG group. Periadventitial administration was performed by ultrasound-guided percutaneous puncture on the first day after angioplasty. Carotid artery specimens were analyzed by immunostaining, Evans blue staining and hematoxylin-eosin staining. ResultsThe RPM particles showed clustered distributions in the FG block. The glue was maintained for a longer time in vivo (> 14 days) than in vitro (approximately 7 days). Two-component liquid FG administered by ultrasound-guided injection completely encapsulated the injured artery before coagulation. The RPM-FG inhibited IH after carotid angioplasty vs. control and other groups. The proliferation of vascular smooth muscle cells (VSMCs) was significantly inhibited during neointima formation, whereas endothelial cell (EC) repair was not affected. ConclusionPeriadventitial delivery of RPM-FG contributed to inhibiting IH in the rat carotid artery injury model without compromising re-endothelialization. Additionally, FG provided a promising platform for the future development of a safe, effective, and minimally invasive perivascular drug delivery method to treat vascular disease.