bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 432: MCUB Regulates Mitochondrial Calcium Uniporter Channel Gating and Modulates Bioenergetics and Cell Death
Authors: Lambert Jonathan P , Luongo TImothy S , Jadiya Pooja , Gao Erhe , Zhang Xueqian , Lucchesee Anna Maria , Elrod John W
Published: July 2021

The mitochondrial calcium uniporter (MCU) is a high-capacity, inward-rectifying channel in the inner mitochondrial membrane and is required for mitochondrial Ca
2+
(
m
Ca
2+
) uptake.
m
Ca
2+
signaling regulates bioenergetics and activates the mitochondrial permeability transition pore (MPTP) which are cellular processes implicated in cardiac pathophysiology warranting further research into the molecular regulation of the MCU. Recently, a
MCU
gene paralog,
MCUB
, was identified as a possible component of the channel. To investigate MCUB’s contribution to uniporter regulation we created a
MCUB
-/-
HeLa cell line using CRISPR-Cas9n. Here, we report that loss of
MCUB
increased
m
Ca
2+
transient amplitude after IP3R stimulation (52% vs. con) suggesting MCUB negatively regulates
m
Ca
2+
uptake. Mitoplast patch-clamping confirmed that loss of MCUB increases MCU current density, suggesting MCUb modulates channel capacitance. Permeabilized

MCUB
-/-

and WT cells exposed to various levels of Ca
2+
(0.5-20μM) revealed that
MCUB
-/-
cells exhibited
m
Ca
2+
uptake at lower Ca
2+
concentrations than controls, suggesting MCUB contributes to channel gating. In
m
Ca
2+
retention capacity experiments
MCUB
-/-
cells required ~30% less bath Ca
2+
to induce depolarization, suggesting a predisposition to
m
Ca
2+
overload. Next, we generated a cardiac-specific, tamoxifen-inducible
MCUB
overexpression mouse model (
MCUB
-Tg). Cardiomyocytes isolated from
MCUB
-Tg hearts exhibited decreased
m
Ca
2+
uptake at low-Ca
2+
(59% vs. con) and isolated mitochondria exhibited a reduction in Ca
2+
-induced swelling (37% vs. con), suggesting a resistance to permeability transition.
MCUB
-Tg mice displayed a significant impairment in isoproterenol-induced contractile reserve and this correlated with a loss of isoproterenol-mediated activation of pyruvate dehydrogenase. In summary, our results suggest that MCUB limits
m
Ca
2+
uptake by altering channel-gating and thereby regulates bioenergetics and MPTP opening.
Abstract 455: The Burmese Python as a Model of Metabolic Protection
Authors: Peter Angela K , Ozeroff Christopher D , Leinwand Leslie A
Published: July 2021

Introduction:
The postprandial Burmese python displays a remarkable level of cardiac adaptation to high levels of circulating triglycerides and an increased metabolic rate without any evidence of cardiac pathology. To challenge the level of cardioprotection, we designed an experiment to attempt to induce metabolic syndrome in the Burmese python. Metabolic syndrome is typically defined as obesity concomitant with other metabolic abnormalities which contribute to an increased risk for cardiovascular disease.

Methods and Results:
Unlike mice or rats who readily undergo hypertrophy in response to pressure-overload or β-adrenergic receptor stimulation, inducing pathological hypertrophy in the Burmese python is challenging. In the current study, we attempted to induce metabolic syndrome through overfeeding. We offered Burmese pythons a meal equivalent to 25% of the python’s body mass every 4 days for a period of 2 months. Two similar groups of pythons were maintained on our standard python feeding schedule (one meal equivalent to 25% of their body weight every 28 days) over the same time course. At the end of 2 months, the weight of the overfed pythons (6197g ± 180g) dramatically increased when compared to fasted (2352g ± 23g) or normal fed pythons (3044g ± 62g). In addition to an overall increase in body weight, the ventricle weight/brain weight ratio increased significantly in the overfed pythons (19.5 ± 1.1) compared to fasted (7.5 ± 0.2) or normal fed pythons (8.8 ± 0.2) indicating considerable hypertrophy. However, despite developing hypertrophy, there was no evidence of aberrant gene expression or impaired glucose tolerance when compared to normal fed pythons.

Conclusions:
Despite repeatedly consuming a large meal, nearly tripling in body weight, and developing significant hypertrophy, the Burmese python is protected from developing metabolic abnormalities such as impaired glucose tolerance. Fetal gene expression markers of pathological cardiac hypertrophy were not induced in overfed pythons. Thus, studying the mechanisms of cardiac adaptation in the Burmese python may potentially provide insights into therapeutic targets for combating metabolic syndrome in patients.
Abstract 456: High Fat Diet-Induced Atherosclerosis-Driven Myocardial Infarction: Role of Cardiac Long Noncoding RNAs in Triiodo-L-Thyronine-Mediated Protection
Authors: Rajagopalan Viswanathan , Schultz Emily , Zhang Youhua , Savinova Olga , Costello Clifford , Yoo Jeanwoo , Domingo Anna , Gerdes A M
Published: July 2021

Novel mechanisms associated with therapeutically safe thyroid hormone (TH) therapy are emerging. We have shown that oral triiodo-L-thyronine (T3) offers safe cardioprotection in coronary ligation myocardial infarction (MI), ligation ischemia-reperfusion injury, diabetic cardiomyopathy, etc. via restoration of gene expression. However, safe therapeutic effects following atherosclerosis-driven MI and role of long noncoding RNAs (lncRNAs) is unknown. We employed a mouse model of scavenger receptor B1 knockout with hypomorphic apolipoprotein E. Young adult heterozygote littermates served as controls and all mice received high fat (HF) diet for one month. Along with HF diet, a cohort of homozygotes (HypoE) received therapeutic dose of T3 (5.5 μg/kg/d) in drinking water ad libitum. In HypoE mice, Paigen HF diet induced interstitial fibrotic MI with severe hypertrophic (Heart wt./Body wt., HW/BW: control:4.6±0.14; HypoE:12.9±0.75; p<0.0001) heart failure, depressed left ventricular (LV) contractility, increased end-diastolic pressure, myocyte disarray/loss, vacuolization and inflammatory cell infiltration. Aortic root showed atheromatous lipid deposits and median survival time was 26 days. Cholate-free paigen HF diet, used to achieve more gradual transition showed moderate hypertrophy (HW/BW: control:4.9±0.1; HypoE:7.9±0.95; ; p<0.01), decreased LV contractility, increasing atrial effective refractory period with a median survival of 41.5 days. Other changes include decreased serum thyroxine, increased serum cholesterol, significant splenomegaly and alterations in real-time gene expression of numerous cardiac lncRNAs and limited serum lncRNAs involved in inflammatory and immune responses (>2-fold; p<0.05). Oral T3 therapy with cholate-free diet partially restored LV contractility, atrial refractory period and cardiac lncRNAs without significantly affecting serum lncRNAs. These were accompanied by expected feedback inhibition of thyroxine without negatively impacting hypertrophy or heart rate. This is the first study to show a novel role of lncRNAs in TH-mediated cardioprotection. It also demonstrates possibility of safe preventive T3 therapy in a clinically relevant early coronary artery disease model.
Abstract 476: Cardiac Adaptation to the Oncometabolite D-2-hydroxyglutarate is Driven by Alpha-Ketoglutarate and Acetyl-CoA
Authors: Karlstaedt Anja , Khanna Radhika , Vasquez Hernan , Taegtmeyer Heinrich
Published: July 2021

Metabolic reprogramming is a hallmark in both cancer and heart failure. Mutations of the isocitrate deyhydrogenase (IDH) 1 and 2 cause metabolic dysfunction in cancer cells through overproduction of the oncometabolite D-2-hydroxyglutarate (D2-HG) and are associated with cardiomyopathy. We recently discovered that alpha-Ketoglutarate dehydrogenase inhibition by D2-HG redirects Krebs cycle flux. This implies a central role for IDH and ATP citrate lyase (ACL) in regulating reductive formation of citrate and histone acetylation in response to mitochondrial impairment in heart and skeletal muscle. Elucidating how metabolic rewiring promotes changes in gene expression and remodeling in heart muscle holds the promise for development of metabolic strategies to support the failing heart. We tested whether modulation of ACL activity reverses D2-HG-mediated metabolic changes using adult rat ventricular cardiomyocytes and L6 myocytes. The ACL inhibitor BMS303141 (BMS) decreased ATP provision in cultured myocytes in a concentration-dependent manner. There was an inverse relation between alpha-KG and ACL activities. Conversely, co-culture with both BMS (0.5 μM) and D2-HG (1 mM) increased ATP provision suggesting that ACL inhibition in presence of D2-HG may be beneficial for energy provision. Next, we conducted isolated working rat heart perfusions with BMS (0.5 μM) and/or D2-HG (1 mM). Cardiac power rapidly declined (by 25%) in the presence of BMS or D2-HG. Simultaneous perfusion with D2-HG and BMS improved cardiac power, suggesting that ACL inhibition protects the heart from metabolic dysfunction by D2-HG. Further, D2-HG elevation mediated structural remodeling in the heart by activating authophagy through increased acetylation of p300, increased phosphorylation of AMPK, and a corresponding decrease in activation and phosphorylation of mTOR. Parallel tracer studies using labeled glucose and glutamine allowed us to conduct computational flux rate analysis by applying the metabolic network CardioNet. We identified major metabolic pathways that are up- and downregulated by D2-HG. Our findings suggest an “oncometabolic axis” in the heart and underscore the potential application of ACL inhibitors to protect the heart from failing.
Abstract 53: Implantation of Loop Recorder In Patients with Ehlers-Danlos Syndrome
Authors: Mistry Hemanshi , Lingampally Sowmika , Butt Amna A , Ashangari Chandralekha , Bhattarai Apeksha , Hamid Stacy E , Suleman Amer
Published: July 2021

Background:
Patients (pts) with Ehlers-Danlos Syndrome (EDS) have a higher incidence of syncope and have a higher rate of complications from incision related procedures. We serve a large population of pts with EDS and report our experience with pts with EDS who required Implantable loop recorder (ILR).

Methods:
We have conducted a retrospective study on a total of 94 pts with ILR implantation from 2014-2016. Out of 94 pts, there were 20 males (average age=50.8
+
20.143 yrs, 21.28%) and 74 females (average age= 47.59
+
17.296 yrs, 78.72%). Total of 24 (25.53%) pts with EDS who had ILR over the last 2 years. Pts were grouped into 3 categories (1) pts with EDS where ILR implantation was done with the standard technique (2) Our learning curve & (3) the results after the standardized new technique for ILR. 1. Category 1 pts, we used the standard provided by the manufacturer to create a V-shaped cut, loop recorder was inserted; subsequently Dermabond was applied. 2. Category 2 pts, we modified the technique, made a more horizontal incision with the V-shaped blade and a 4-0 Vicryl suture was used to improve wound healing. 3. Category 3 pts, after standardizing the technique, we used the V-shaped blade provided with 4-0 Vicryl knotless continuous suture to tamponade the bleeding. The Dermabond was applied only after local hemostasis was observed.

Findings:
When the loop recorder was implanted using the standard technique, more of bleeding, bigger scars and multiple keloid formation was observed. In the category 1, we learned that if proper hemostasis is not obtained during the implantation, the patient tended to have more scars. In category 3, when 1-2 layers of Vicryl sutures were applied, the first layer of sutures were subcutaneous & the second was under the skin and a knot less closure was done, hemostasis was obtained prior to applying Dermabond, patients did not have scar formation.

Conclusions:
We hereby conclude that EDS pts who required ILR, the wound should be closed with 1-2 layers of Vicryl using a knot-less closure technique which will leave a more aesthetically acceptable scar. We would also recommend that until further research is done, this same technique should be use in patients with other hereditary connective tissue disorders.
Abstract 60: Inducible Arrythmogenicity of Direct Current Electroporation Ablation: Insight from a Series of Acute Canine Studies
Authors: Sugrue Alan , Witt Chance , DeSimone Christopher V , Padnanabhan Deepak , Killu Ammar , Naksuk Niyada , Kapa Suraj , Asirvatham Samuel J
Published: July 2021

Background:
The use of direct current electroporation has the potential for significant utility because of its non-thermal approach to tissue destruction. However, the fear of inducibility of cardiac arrhythmias (particular ventricular fibrillation) when using electroporation remains of concern due to membrane poration and ion flux during periods of vulnerability occurring in ventricular repolarization.

Objectives:
Critically examine the incidence of arrhythmias in a series of acute canine studies to retrospectively determine cause and safe electoporative dosing margins.

Methods:
We performed electroporation ablation in 6 acute canine studies. These were experimental studies performed at sites critical in arrhytmogenesis. Sites included the pulmonary veins, left atrial appendage, superior vena cavae, right atrium and ventricle. Electroporation was delivered using an ECG gating algorithm so that QRS complexes are tagged and direct current energy is not delivered during the vulnerable portion of the T wave.

Results:
In 6 acute canine experiments, we delivered a total of 62 electroporation applications for ablation purposes. The average electroporation dosage delivered involved an average of 1427 Volts (range 750-3000 V), Pulse length of 100 ms, and number of pulses of 20.2 (range 10-100). AF was induced in 27.4% of electroporiatve applications. Atrial flutter/tachycardia occurred in 8.1%. VF occurred in only one application at a location of the left superior pulmonary vein.

Conclusion:
These data suggest that induction of VF is relatively uncommon with ECG gating and highlight its importance when using this modality. However, the induction of AF occurs with higher frequency. The actual mechanism as to why this occurs requires further systematic study.
Abstract 72: Human Vascular Smooth Muscle Gene Expression Profiling Between Incracranial and Extracranial Vasculature
Authors: Kim Hyung-Seok , Park Jong Tae , Na Joo Young , Park Man Seok
Published: July 2021

Background:
Intracranial arteries (IA) have unique histological, pharmacological, and pathological characteristics when compared to extracranial arteris (EA). Despite its importance, little is known about the molecular characteristics of those vessels. In this study, we carried out a genome-wide characterization on expressions of mRNAs, and performed integrative analyses in conjunction with changes of the transcriptome between IA and EA vascular smooth muscle cells.

Methods:
mRNA expression screening was performed from same autopsied body (n=3) using Agilent Human mRNA arrays for IA (middle cerebral a.) and EA (left ant. descending a. of coronary a.) vascular smooth muscle (vSMC) layer. Total gene expression compared with intracranial vs. extracranial vSMC.

Results:
As compared to EA, we identified 304 mRNA that were differentially expressed in the IA vSMC (p<0.05 and fold change >2), including 224 upregulated and 80 downregulated. Through this comparison we have identified angiogenesis, cell migration, cell proliferation, neurogenesis, and inflammatory response genes whose expression is enriched in IA vSMC. This analysis has identified that RAR activation, and Caveolar-mediated endocytosis signaling cascade is specifically enriched at the IA vSMC, implicating this pathway in regulating this arterial system.

Conclusions:
This dataset provides a resource for understanding the different arterial regulation and disease susceptibility in IA and EA, especially atherosclerosis and diabetes.Funding: This study was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Kim HS, NRF-2016R1A2B4008316).
Abstract 73: Pre- and Post-Translational Regulation of Lysyl Oxidase/β1-Intergrin in Aortic Vascular Smooth Cell of Hypertension
Authors: Ma Ben , Zhou Ning , Stoll Shaunrick , Qiu Hongyu
Published: July 2021

Background:
An increase in aortic stiffness is a fundamental component of hypertension
.
We previous showed that aortic vascular smooth muscle cells (VSMCs) play an causative role in aortic stiffening not only due to the increased intrinsic stiffness in VSMC itself but also due to its interaction with extracellular matrix (ECM). However, the molecular mechanism involved is unclear. Here, we test the hypothesis that the aortic VSMCs mediates pre- and post-translational regulation of Lysyl Oxidase (Lox)/β1intergrain pathway which may be one of the mechanisms of VSMCs-ECM remodeling in hypertension.

Methods and Results:
4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured with an invasive catheter and Doppler imaging echocardiography, respectively. Compared to WKY, SHR showed a significant higher aortic pressure (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, (
p
<0.01) and a greater aorta stiffness reflected by 2.5-fold higher arterial stiffness index (ASI) in SHR vs WKY (
p
<0.01). VSMCs were isolated from thoracic aorta, then cultured and measured at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Compared to WKY, VSMCs from SHR showed a significant increase in mRNA Lox, a copper-dependent amine oxidase mediating the crosslinking of collagens and elastin (2.7 folds vs WKY,
p
<0.01). The abundance of LOX protein was significant increased not only at pro-enzyme lox level, but also at the regulatory pro-peptide lox and matured active enzyme Lox in SHR VSMCs by 3.5 folds and 1.4 folds respectively (
p
<0.01), indicating an increased post-translational regulation of LOX in SHR VSMCs. In addition, the glycosylated β1 integrin, a transmembrane receptor that facilitate cell-ECM adhesion, was found to be significant increase in SHR VSMCs vs WKY.

Conclusion:
The regulation of pre- and post-translational regulation of Lox/β1intergrain pathway in aortic VSMCs presents a novel mechanism of cell-ECM interaction, which may contribute to the increased aortic stiffness in SHR.
Abstract 88: A Crucial Role of BAG3 in Preventing Dilated Cardiomyopathy
Authors: Fang Xi , Bogomolovas Julius , Zhang Wei , Wu Tongbin , Liu Canzhao , Lowe Jennifer , Ouyang Kunfu , Zhang Zhiyuan , Ma Xiaolong , Mu Yongxin , Stroud Matthew , Lao Dieu-Hung , Dalton Nancy , Gu Yusu , Wang Celine , Wang Michael , Liang Yan , Lange Stephan , Peterson Kirk , Evans Sylvia , Chen Ju
Published: July 2021

Defective protein quality control (PQC) systems are implicated in multiple diseases, with molecular chaperones/co-chaperones being critical to PQC. Cardiomyocytes are constantly challenged by mechanical and metabolic stress, placing great demand on the PQC system. Mutations and downregulation of the co-chaperone protein
B
cl-2-
a
ssociated athano
g
ene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to Dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and mechanisms by which the E455K mutation lead to DCM remained obscure. Here, we found that cardiac-specific BAG3 knockout (CKO) and cardiac-specific E455K BAG3 knockin mice developed DCM. Comparable phenotypes in the two mutants demonstrated that the E455K mutation resulted in loss-of-function, and experiments revealed that the E455K mutation disrupted interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a specific subset of proteins required for metabolic and contractile function of cardiomyocytes was enriched in the insoluble fraction. Together, these observations suggested that interaction between BAG3 and HSP70 was essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide new insight into the pathogenesis of heart failure caused by defects in BAG3 pathways, suggesting that increasing protein levels of BAG3 may be of therapeutic benefit in heart failure.
Abstract 98: Canonical TGF-β1 Signaling in Cardiomyocytes is Essential to Maintain Basal Cardiac Function
Authors: Umbarkar Prachi , Singh Anand P , Verma Vipin K , Gupte Manisha , Guo Yuanjun , Zhang Qinkun , Force Thomas L , Lal Hind
Published: July 2021

The role of canonical transforming growth factor-β (TGF-β) pathway is well recognized in fibroblast biology and fibrosis in diseased hearts. However, its role in cardiomyocyte (CM) biology is not clear. SMAD4 is the central intracellular mediator of canonical TGF-β signaling. Herein, we investigate the role of SMAD4 in CM-biology and cardiac pathophysiology. SMAD4 homozygous floxed (

SMAD4
fl/fl

) and heterozygous floxed (

SMAD4
fl/-

) mice were crossed with α-myosin heavy chain (Mer-Cre-Mer) to create conditional CM-specific SMAD4-KO (

SMAD4
fl/fl
Cre
+/-

) and SMAD4 haploinsufficiency (

SMAD4
fl/-
Cre
+/-
),

respectively. At 10 Wks of age, mice were subjected to well established tamoxifen diet protocol for 2 Wks. Echocardiographic analysis at 4 Wks post-tamoxifen treatment reveals that CM-specific loss of SMAD4 leads to dilatative ventricular remodeling as reflected by significantly increased LVIDs. This dilatative remodeling was associated with severe ventricular dysfunction as reflected by significantly reduced ejection fraction (EF) and fractional shortening (FS). Both HW/BW and LW/BW were significantly elevated in SMAD4 KO mice, suggesting pathological hypertrophy and heart failure in KOs. Analysis of Masson trichrome stained heart sections reveals a marked increase in fibrosis in the KO hearts. Q-PCR analysis showed the re-expression of fetal gene program (ANP, BNP), further confirming pathological remodeling in the KO hearts. As the heart functions of heterozygous mice and littermate controls were comparable at baseline, we stress them with TAC surgery. Consistent with KO findings, at 4 Wks post-TAC, heterozygous mice demonstrated characteristic heart failure phenotype. Taken together, these findings suggest that SMAD4 is required to maintain basal cardiac function and to prevent adverse ventricular remodeling in a pressure-overloaded heart.
Calcium and Vascular Smooth Muscle Contraction
Authors: SEIDEL CHARLES L. , BOHR DAVID F.
Published: July 2021

Radioactive calcium flux in rabbit aorta was studied to evaluate factors regulating the movement of calcium into and out of the cell. Calcium content was estimated both from (1) the amount of
45
Ca present in the tissue and the specific activity of Ca in the bathing medium, and (2) determination of chemical calcium by atomic absorption spectrophotometry. Calcium efflux was followed by washing aortic strips previously loaded with
45
Ca in nonlabeled salt solution.

Norepinephrine (10
-6
g/ml) caused a significant increase in the calcium content calculated from radioactive calcium activity, whereas isoproterenol (10
-8
g/ml) caused a significant decrease in calcium content. Measurements of efflux rates, first in calcium-free solution and subsequently in 1.6 m
M
Ca, demonstrated that norepinephrine delays calcium efflux into calcium-free solution and delays the rapid exchange of calcium that occurs upon exposure of muscle to a calcium-containing salt solution. Isoproterenol produced no significant change in these rates. These observations are compatible with the possibilities that norepinephrine causes contraction by decreasing calcium efflux and that isoproterenol causes relaxation by decreasing calcium influx.
CT Perfusion Imaging versus MR Diffusion Weighted Imaging: Prediction of Final Infarct Size in Hyperacute Stroke
Authors: Berzin Tyler M , Lev Michael H , Goodman Daniel , Koroshetz Walter J , Hunter George J , Hamberg Leena , Buonanno Ferdinand , Putman Christopher , Budzik Ronald , Eskey Clifford , Schwamm Lee H , Gonzalez Ramon G
Published: July 2021

10

Background:
Both CT perfusion imaging (CTP) and MR diffusion weighted imaging (DWI) have been applied clinically to predict tissue outcome in patients with hyperacute stroke, however these techniques have not been directly compared in the same patient population. In CTP, whole brain CT scanning is performed during the steady state administration of a contrast bolus, creating both perfused blood volume weighted and CT angiographic images. Purpose: To determine the value of CTP imaging, compared to DWI, in predicting final infarct size in hyperacute stroke patients. Materials and
Methods:
CTP followed by DWI imaging was performed in 23 consecutive patients presenting within 12 hours of stroke onset. Mean time from stroke onset to imaging was 4.6 hours for CTP, 5.4 hours for DWI, and 6.5 days for conventional nonenhanced CT and/or MR follow-up. No patients had secondary events between initial and follow-up scanning. Initial and follow-up ischemic volumes were computed from the CTP, DWI, and follow-up scans using image segmentation software, and compared using linear and multiple regression.
Results:
CTP and DWI volumes were independent predictors of final infarct size (p=0.02). The regression line for CTP volume vs. final infarct size had slope 1.02 and r
2
= 0.88 (p<0.0001). The regression line for DWI volume vs. final infarct size had slope 1.41 and r
2
= 0.92 (p<0.0001). Overall sensitivity and specificity for parenchymal stroke
detection
were 83% and 100% for CTP, and 100% and 100% for DWI, respectively.
Conclusion:
Although DWI is more sensitive than CTP for parenchymal stroke detection, both DWI and CTP are highly accurate predictors of final infarct volume. DWI tends to underestimate final infarct size, whereas CTP more closely approximates final infarct size.
Temporal and spatial expression of neuropilin-1 in focal cerebral ischemic brain
Authors: Zhang Zheng G , Tsang Wayne , Zhang Li , Powers Cecylia , Chopp Michael
Published: July 2021

6

Neuropilin-1 is a receptor for vascular endothelial grow factor 165 isoform (VEGF
165
) and plays roles in vasculogenesis and angiogenesis. Neuropilin-1 also binds to semaphorin III which is an inhibitor to axon guidance signal. To seek evidence that neuropilin-1 may play roles in ischemic brain, we examined the temporal and spatial profiles of its expression after focal cerebral ischemia. Male Wistar Rats (n=42) were subjected to 1h to 28 days of embolic middle cerebral artery (MCA) occlusion. Expression of neuropilin-1 was measured by Northern blot, in situ hybridization and immunohistochemistry. Upregulation of neuropilin-1 mRNA was detected in the ischemic hemisphere on Northern blots at 2h and persisted at least 28 days after ischemia. In situ hybridization revealed increased mRNA in the ischemic injury neurons around the boundary of the ischemic lesion at 4h to 48h after ischemia. Seven to twenty-eight days after ischemia, neuropilin-1 mRNA was localized to vessels within the ischemic lesion. Immunostaining for neuropilin-1 showed that some of the ischemic neurons were neuropilin-1 immunoreactive at 2h to 4h after ischemia, and cerebral blood vessels within the ischemic lesion were neuropilin-1 immunoreactive at 2 days to 28 days after ischemia. In addition, hypertrophic astrocytes around the boundary of the ischemic lesion were neuropilin-1 immunoreactive at 1 day to 28 days after ischemia. Double immunofluorescent staining for neuropilin-1 and VEGF showed that neuropilin-1 immunoreactive vessels and astrocytes exhibited VEGF immunoreactivity. Thus our data suggest that upregulation of neuropilin-1 may play a role in angiogenesis in ischemic brain.
EPAR Therapy System for Treatment of Acute Stroke: Safety Study Results
Authors: Lutsep Helmi L , Campbell Morgan , Clark Wayne M , Jansen Olav , Lefkowitz Michael A , Marks Michael P , Norbash Alex , Putman Chris , Schumacher Martin
Published: July 2021

19

Background.
Laser thrombolysis offers the potential for rapid clot removal. This multicenter study assessed the safety of the Endovascular Photo Acoustic Recanalization (EPAR) laser system in the intra-arterial treatment of acute stroke.
Methods.
Patients had a NIHSS score of 4 or more and angiographically demonstrated occlusion (TIMI 0–1) of the internal carotid (ICA), anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), basilar or vertebral artery. Patients were treated within 6 hrs of stroke in the anterior circulation and within 24 hrs in the posterior circulation. Cerebral blood flow before and after the EPAR procedure was assessed angiographically. Clinical outcome was measured at baseline, 7 days and at 30 days using the Modified Rankin (MR) and NIHSS. Both power and energy were adjusted upwards during the trial to establish the limits of safety.
Results.
Twenty-seven patients were enrolled in the study and 26 patients were treated with the laser. The baseline median NIHSS score was 21 (range 12–40) and the mean age was 65 (range 39–78). The vessels treated included four supraclinoid ICAs, one ACA, 16 MCAs, two PCAs, seven basilars and one vertebral artery. Twelve patients had died by 30 days (46%). Five patients developed a symptomatic ICH (19%), four of whom also received lytics. No complications occurred during active lasing. Three vessel perforations occurred, one prior to introduction of the EPAR device and two during manipulation of the wire and device prior to lasing. None of these extravasations was associated with any untoward clinical effects. Of 31 vessel segments treated, fifteen (48%) improved by at least one grade of TIMI flow after EPAR laser treatment. In those patients who survived, the median NIHSS score was 17 (range 2–38) at 7 days (n=16) and 15 (range 1–37) at 30 days (n=12). The mean MR was 4.3 (range 2–5) and 3.5 (range 0–5) at 7 and 30 days, respectively.
Conclusions.
The EPAR laser system has shown acceptable safety, demonstrating no serious complications during active lasing. The high mortality in this population is comparable to that in other groups with similarly severe presenting strokes. Further clinical studies are planned.
Genetic and Environmental Risk Factors for Intracerebral Hemorrhage
Authors: Woo Daniel , Sauerbeck Laura , Kissela Brett M , Khoury Jane C , Shukla Rakesh , Moomaw Charlie , Graus Jessica A , Carrozzella Janice , Jauch Edward C , Pancioli Arthur M , Broderick Joseph P
Published: July 2021

27

Introduction:
We report a planned midpoint analysis of a prospective, population-based, case-control study of the genetic and environmental risk factors of spontaneous, non-traumatic, intracerebral hemorrhage (ICH).
Methods:
Cases were matched to two controls by age, race and gender. Data was obtained by direct interview and review of all available medical and neuroimaging data. Apolipoprotein E (Apo E)genotype was determined by polymerase chain reaction. Multivariable analyses were performed using logistic regression modeling.
Results:
Between 6/97 and 2/00, 189 cases of ICH (150 white/39 black; 68 lobar/121 non-lobar) and 368 controls were enrolled into the study. Independent risk factors for multivariable analysis are listed in the table. Only prior stroke was an independent risk factor for both lobar and non-lobar ICH.
Conclusions:
The importance of individual genetic and environmental risk factors for ICH vary substantially by location of ICH. A history of a first-degree relative with ICH was associated with an increased risk of lobar ICH, independent of Apo E genotype. This finding indicates that other genetic risk factors may be important in the development of ICH.
Duration of Arterial Recanalization During Intravenous TPA Therapy: Classification and Short-term Improvement
Authors: Alexandrov Andrei V , Demchuk Andrew M , Burgin W Scott , El-Mitwalli Ashraf , Grotta James C
Published: July 2021

47

Background:
Arterial recanalization precedes clinical improvement in ischemic stroke. However sudden reperfusion may increase the chance of edema or hemorrhage.
Methods:
Consecutive patients given intravenous TPA were monitored using transcranial Doppler (TCD). The NIH Stroke Scale (NIHSS) was performed before bolus and at 24 hours. Patients with complete or partial recanalization according to validated criteria were analysed. Duration of recanalization was classified a priori as
sudden
(abrupt appearance of a normal or stenotic low resistance signal),
stepwise
(flow improvement over 1–29 minutes), and
slow
(30–60 minutes).
Results:
Recanalization occurred in 43 patients (age 68±17 years, NIHSS 16.8±6, median 15 points, TPA bolus at 135±61 minutes after onset). Recanalization began at 23±19 min (median 17) and completed at 42±23 min (median 35) after bolus. Duration of recanalization was 23±16 min (median 17 min). Recanalization was sudden in 5, stepwise in 23, and slow in 14 patients. Faster recanalization predicted better short term improvement (Table, p=0.03). Symptomatic hemorrhage occurred in 1 patient with stepwise recanalization at 5.5 hours after onset. Slow and/or partial recanalization with dampened flow signal was found in 53% of patients with NIHSS ≥ 10 points at 24 hours (p=0.01).
Conclusions:
Short duration of recanalization is associated with better short-term improvement mostly likely due to faster clot break-up and low resistance of the distal circulatory bed. Slow flow improvement and dampened flow signal are poor prognostic signs that may be useful in patient selection for interventional treatment.
A reliable new method to estimate >1/3 middle cerebral artery infarction on early computed tomography scan
Authors: Silver Brian , Tamayo Arturo , Wong Edward H , Demaerschalk Bart , Merino Jose G , Hachinski Vladimir
Published: July 2021

50

Objectives:
To develop a reliable method of estimation of >1/3 involvement of the middle cerebral artery territory (MCAT) on early CT scan.
Background:
The European Cooperative Acute Stroke Study (ECASS) suggested that > 1/3 involvement of the MCAT on early CT scan was a risk factor for symptomatic intracerebral hemorrhage (SICH) following treatment with tissue plasminogen activator (tPA) for acute ischemic stroke (AIS). There are no published guidelines on how to perform such an evaluation. The ICE method was developed for the purpose of standardizing the approach of infarct size estimation. This method requires the observer of the CT scan to mentally: 1) Idealize the MCAT into an area approximating a trapezoid, 2) Close a geometric figure around the area(s) of hypodensity(ies), and 3) Estimate the ratio of the two geometric figures (C/I).
Methods:
Five stroke fellows (observers) were tested with 40 CT scans which had been performed within the first few hours of an acute neurological event. Observers were blinded to patient history. They were asked whether to treat the patient on the basis of the CT scan. The decision not to treat was predicated on the presence of hemorrhage or > 1/3 MCAT involvement.
Results:
Five scans showed evidence of hemorrhage. All observers detected the hemorrhages. The remaining scans were of patients with presumed ischemia. Six scans were thought to show > 1/3 MCAT involvement by all observers. For all scans, median interobserver agreement among 10 paired comparisons was 86% (range 73–93%). Median kappa was 0.72 (range 0.44–0.85, p<0.001).
Conclusion:
The ICE method has very good interobserver reliability. The method merits further investigation among non-stroke physicians.
Bcl-2 Overexpression Protects Against Neuron Loss in Peri-infarct Regions Following Experimental Stroke
Authors: Zhao Heng , Yenari Midori A , Lawrence Matthew S , Chen Danye , Ho Dora Y , Sapolsky Robert M , Steinberg Gary K
Published: July 2021

59

Purpose:
Bcl-2 protects against both apoptotic and necrotic death induced by various insults, including cerebral ischemia. Ischemic injury with and without reperfusion may occur by different mechanisms, and protective strategies may be effective against one, but not the other. Here, we determine whether Bcl-2 protects against 2 different forms of focal cerebral ischemic injury within cortical penumbra in a model of experimental stroke.
Methods:
Bipromoter defective herpes simplex viral (HSV) vectors expressing Bcl-2 plus β-galactosidase (β-gal) as the reporter or β-gal only (control vector)were stereotaxically injected into left and right cortices using coordinates corresponding to the infarct border. 12 h later, focal ischemia was induced by occluding the middle cerebral artery (MCA) and both common carotid arteries (CCA). For the permanent occlusion model, the MCA was left occluded, but the CCAs were reopened after 2 h. For the reperfusion model, all 3 vessels were reopened after 2 h. 2 days later, brains were harvested and sections were reacted with X-gal followed by cresyl violet. X-gal positive neurons were counted in each hemisphere and expressed as the ratio of positive neurons in the ischemic side compared to the non-ischemic side.
Results:
Bcl-2 overexpression resulted in significantly improved cortical neuron survival within penumbral regions compared to control vector in both cases. Following reperfusion, Bcl-2 injected animals had 74±14%(n=5) survival compared to control 26±3% (n=6)(P<0.05). Following permanent MCA occlusion, Bcl-2 injected animals had 92±9%(n=5)survival vs.control, 45±5%(n=7)(P<0.05). The extent of survival from the 2 different paradigms suggest that Bcl-2 mediated protection was greater in the ischemia with permanent occlusion compared to reperfusion group.
Conclusion:
Bcl-2 overexpression protects against experimental stroke with and without reperfusion. The extent of protection appears to be greater for ischemia with permanent MCA occlusion. Furthermore, we show that gene transfer to penumbral zones is possible, suggesting a potential therapeutic strategy for clinical application.
NXY-059, the free radical trapping nitrone-based agent, reduces hemiparesis and reduces grey and white matter damage after focal cerebral ischemia in monkeys
Authors: Marshall J Wb , Duffin K J , Green A R , Ridley R M
Published: July 2021

60

There is little published evidence for protection of white matter with neuroprotective drugs in animal models of stroke, yet white matter protection may be important in achieving clinical efficacy. We have examined the effects of NXY-059, a nitrone-based free radical trapping agent, on long-term functional disability in a primate model of stroke. We also examined histopathological effects, including analyses of grey and white matter damage. Five minutes after unilateral permanent middle cerebral artery occlusion, marmosets received a 1 ml i.v. infusion of saline (n=5) or NXY-059 (28 mg/kg) (n=6) and osmotic minipumps (model 2001D) were implanted s.c. to provide continuous drug or saline infusion for 48 h. Drug-filled pumps released NXY-059 at a rate of approximately 16 mg/kg/h. The plasma unbound drug concentration at 24 h was 76.3 ± 5.7 μM, a level well tolerated in acute stroke patients. The monkeys had been trained and tested on a variety of behavioral tasks before surgery. NXY-059-treated monkeys were significantly better at reaching with their contralesional arm than were saline-treated monkeys when re-tested 3 (p<0.01) and 10 weeks (p<0.01) after surgery. NXY-059-treatment also significantly reduced spatial neglect measured 3 weeks after surgery (p<0.01) compared with the saline group. After behavioral testing was complete, histopathological analysis showed NXY-059-treated monkeys had significantly smaller infarcts than saline-treated monkeys (F (1, 10)=5.21, p<0.05). NXY-059 reduced overall infarct size by 51%, damage to the cortex was reduced by 54%, white matter by 52%, caudate by 49%, and putamen by 33% compared with saline-treated monkeys. In conclusion, NXY-059 substantially lessened the functional disability in these monkeys. This drug protects not only cortical tissue, but also white matter and subcortical structures against ischemic damage. Together these findings bode well for advancing this drug to further clinical trials for its use in acute stroke.
National Rates of Warfarin Use in Medicare Patients with Atrial Fibrillation: Results from the National Stroke Project
Authors: Nilasena David S , Kresowik Timothy F , Piskac Anton F , Hemann Rebecca A , Brenton Marian A , Wilwert June M , Hendel Marc E , Nevins Lynnette E
Published: July 2021

66

Background:
The National Stroke Project is a HCFA initiative to improve stroke related hospital care for Medicare beneficiaries. As part of the evaluation of these efforts, HCFA is measuring performance on a set of quality indicators at the state and national levels. We report the baseline results for a key quality indicator for this project: warfarin at discharge for patients with atrial fibrillation (AF).
Methods:
Project data were abstracted from a national sample of Medicare inpatient charts with a principal or secondary diagnosis of AF (ICD-9-CM 427.31) and discharged between 4/98 and 3/99. All U.S. states, the District of Columbia, and Puerto Rico were sampled using a systematic random approach. Eligible patients were required to have physician confirmation of AF during the hospitalization and at discharge, or intermittent AF. The main outcome measure was a prescription or physician documented plan for warfarin at discharge.
Results:
Of 38,925 cases reviewed, 12,303 met the inclusion criteria. Many of the cases (38.3%) were excluded due to a history or current finding of hemorrhage. Nationwide, 6,633 (unadjusted rate, 53.9%) patients were prescribed warfarin at discharge or had a plan to start warfarin after discharge. The state-specific rates ranged from 30.7% to 65.3%. Univariate analyses showed that warfarin was prescribed less frequently (p<0.002) for adults 85 years of age and older (rate=39.9%, OR=0.47, 95% CI=0.43–0.51) and women (rate=52.0%, OR=0.83, 95% CI=0.78–0.90). African-Americans (rate=47.2%, OR=0.76 95% CI=0.63–0.90) and Asians (n=108, rate=37.0%, OR=0.50, 95% CI=0.34–0.74) were also found to have lower warfarin therapy rates.
Conclusions:
The results from this large national sample confirm the findings from other reports that there is substantial under-utilization of warfarin therapy for stroke prevention among Medicare patients with AF. This is particularly true for demographic subgroups at high risk for stroke. Quality improvement efforts are currently underway through HCFA’s National Stroke Project (AF topic) to increase warfarin use in appropriate AF patients.
TRIFLUSAL VERSUS ASPIRIN IN SECONDARY STROKE PREVENTION:RESULTS OF TACIP STUDY
Authors: Matias-Guiu Jordi , Ferro Jose , Alvarez-Sabin Jose , Torres Ferran
Published: July 2021

73

TACIP was a multicentre, double-blind, randomised, parallel trial comparing the efficacy and safety of triflusal (600 mg/od) and ASA (325 mg/od) in patients who had a recent TIA or non disabling stroke. The primary end-point was the combined occurrence of non-fatal stroke, non-fatal AMI or cardiovascular death. Any of them separately was a secondary end-point, as well as total mortality, major systemic or cerebral hemorrhage and systemic thromboembolism. Forty-three centres in Spain and Portugal participated in the trial. A total of 2108 patients were evaluated with a mean follow-up of 31 months. There were no significant differences between both groups regarding to baseline characteristics. Main results were: The incidence of major and minor hemorrhages were higher in ASA than in triflusal group (24.5% vs 16.4%; p<0.001).
Conclusion:
Triflusal and low-dose ASA have a similar efficacy in secondary stroke prevention. Major and minor hemorrhages were significantly reduced by triflusal. Triflusal is a safer alternative to aspirin in the secondary prevention of stroke.
Redo Carotid Endarterectomy Versus Primary Carotid Endarterectomy
Authors: AbuRahma Ali F , Jennings Tucker G , Wulu John T , Tarakji Lisa , Robinson Patrick A
Published: July 2021

90

Background/Purpose:
Several authorities have recently advocated carotid stenting for recurrent carotid stenosis because of the perception that redo surgery carries a higher complication rate than primary carotid endarterectomy (CEA). This study will compare early and late results of reoperations versus primary CEA. Patient Poplulation and
Methods:
All redo operations for recurrent carotid stenosis performed during a recent 7-year period by a single vascular surgeon were compared with primary CEA. Since all redo CEAs were done using polytetrafluoroethylene (PTFE) or vein patch closure, only primary CEAs using the same patching were analyzed. A Kaplan Meier life-table analysis was used to estimate stroke-free survival rates and freedom from ≥50% recurrent stenosis.
Results:
Out of 510 primary CEAs, 265 had PTFE or vein patch closure. One hundred twenty-four reoperations using PTFE or vein patch closure were done during the same period. Both groups had similar demographic characteristics. Indications for reoperations and primary CEAs were symptomatic stenosis in 78% and 58%, and asymptomatic ≥80% stenosis in 22% and 42%, respectively (p<0.001). The 30-day perioperative stroke and transient ischemic attack rates for reoperation and primary CEA were 4.8% versus 0.8% (p=0.015) and 4% versus 1.1%, respectively, with no perioperative deaths in either group. Cranial nerve injury was noted in 17% in reoperation patients versus 5.3% in primary CEA patients, however most of these were transient (p<0.001). The mean hospital stay was 1.8 days for reoperation versus 1.6 days for primary CEA. The cumulative stroke-free survival and freedom from ≥50% recurrent stenosis rates for reoperation at 1, 3, and 5 years were 96%, 91%, 82%, and 98%, 96%, 95%, respectively; and 94%, 92%, 91% and 98%, 96%, 96%, respectively for primary CEA (no statistically significant differences).
Conclusions:
Reoperation carries higher perioperative stroke and cranial nerve injury rates than primary CEA. However, redo operations are durable and have stroke-free survival rates that are similar to primary CEA. These considerations should be kept in mind when recommending carotid stenting versus reoperation.
The Association of Carotid Artery Intimal-Medial Thickness and Allelic Variants of Stromelysin-1, Interleukin-6 and Hepatic Lipase Genes: A Pilot Study from the Northern Manhattan Stroke Study
Authors: Rundek Tanja , Elkind Mitchell S , Boden-Albala Bernadette , Humphries Steve E , Sacco Ralph L
Published: July 2021

111

Objective:
To determine the association between carotid artery intimal-medial thickness (IMT) and functional promoter variants of stromelysin-1 (
MMP3
: -16125A>6A), interleukin-6 (
IL6
: -174G>C) and hepatic lipase (
HL
: -480C>T) genes.
Background:
Carotid artery intimal-medial thickness (IMT) correlates well with pathologically defined atherosclerosis, a complex multifactorial disorder with both hereditary and environmental causes. IMT is also a risk factor for stroke, myocardial infarction and death.
Methods:
High-resolution B-mode imaging and mean intimal-medial thickness (IMT) measurements in common carotid arteries were obtained in 87 subjects (mean age 70±12 years; 55% women; race-ethnicity: 60% Hispanic, 35% black, 13% white) using a standardized carotid artery IMT protocol. Genotyping was assessed using PCR amplification and restriction digestion with enzymes specific to the alleles.
Results:
For all polymorphisms genotype distribution was as expected from Hardy-Weinberg proportions. The frequencies of the rare alleles at each locus in the sample were:
MMP3
5A 0.32 (95% CI, 0.25–0.39),
IL6
-174C 0.19 (95% CI, 0.13–0.25) and
HL
-480T 0.42 (95% CI, 0.35–0.50). For
MMP3
and
IL6,
but not
HL,
these frequencies were significantly lower than reported in Caucasian subjects. The overall mean common carotid artery IMT was 0.86±0.20 mm. Subjects with the genotypes
MMP3
6A6A or
HL
CC had 8% greater mean common carotid artery IMT than the other genotypes combined (
MMP3
6A6A: 0.93 mm vs. 0.86 mm, absolute IMT difference =0.07 mm;
HL
CC: 0.93 mm vs. 0.86 mm, absolute IMT difference = 0.07 mm). Subjects with
IL6
GG had 2% higher common carotid artery IMT compared to others combined (
IL6
GG: 0.89 mm vs. 0.87 mm, absolute IMT difference).
Conclusion:
We observed higher common carotid artery intimal-medial thickness in subjects homozygous for specific candidate genes related to matrix deposition, lipid metabolism and inflammation. Further studies with increased power are needed to confirm these genotype-phenotipe associations.