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Background: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. Methods: We utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia. Results: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP + CD144 + CD11b − fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP + CD11b - cells from the plug revealed a small subset of YFP + CD144 + CD11b − E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. Conclusions: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.

Heart failure is characterized by pathologic hemodynamic derangements, including elevated cardiac filling pressures ("backward" failure), which may or may not coexist with reduced cardiac output ("forward" failure). Even when normal during unstressed conditions such as rest, hemodynamics classically become abnormal during stressors such as exercise in patients with heart failure. This has important upstream and downstream effects on multiple organ systems, particularly with respect to the lungs and kidneys. Hemodynamic abnormalities in heart failure are affected by processes that extend well beyond the cardiac myocyte, including important roles for pericardial constraint, ventricular interaction, and altered venous capacity. Hemodynamic perturbations have widespread effects across multiple heart failure phenotypes, ranging from reduced to preserved ejection fraction, acute to chronic disease, and cardiogenic shock to preserved perfusion states. In the lung, hemodynamic derangements lead to the development of abnormalities in ventilatory control and efficiency, pulmonary congestion, capillary stress failure, and eventually pulmonary vascular disease. In the kidney, hemodynamic perturbations lead to sodium and water retention and worsening renal function. Improved understanding of the mechanisms by which altered hemodynamics in heart failure affect the lungs and kidneys is needed in order to design novel strategies to improve clinical outcomes. Dr Verbrugge is supported by a Fellowship of the Belgian American Educational Foundation and by the Special Research Fund of Hasselt University (grant no. BOF19PD04). Dr Borlaug is supported by grants R01 HL128526 and U01 HL125205, both from the US National Heart, Lung, and Blood Institute. Borlaug, BA (corresponding author), Mayo Clin & Mayo Fdn, 200 First St SW, Rochester, MN 55905 USA. borlaug.barry@mayo.edu

Introduction: Cardiac and multiple organ network is involved in the pathophysiology of heart failure (HF). Hepatokines, which are produced and secreted from the liver, have regulatory functions in the peripheral tissues. However, the interaction between the heart and liver or clinical relevance of hepakokines in HF remains poorly understood. The present study aimed to elucidate the clinical significance of hepatokine selenoprotein P in HF. Methods and Results: We enrolled 250 participants including 171 hospitalized patients with HF and 79 control subjects. The serum concentration of selenoprotein P was measured by an enzyme-linked immunosorbent assay. We found that the serum selenoprotein P levels in HF patients were significantly higher than those in controls (14.8 ± 7.5 vs. 8.4 ± 4.6 mg/L, P1.33 m/s) and liver hypoperfusion by peak systolic velocity of the celiac artery (celiac-PSV, Figure A ). Selenoprotein P was negatively correlated with celiac-PSV (PFigure B ). Conclusion: Upregulation of liver-derived selenoprotein P was associated with hepatic hypoperfusion, not hepatic congestion, and related to the adverse prognosis in HF. Selenoprotein P may be a novel molecule involved in the cardio-hepatic interaction.

Introduction: We developed a technique for Stage 1 “Norwood” reconstruction with continuous perfusion of the head, heart, and lower body at mild hypothermia, deemed Sustained Total All-Region (STAR) perfusion. Hypothesis: We hypothesized that STAR perfusion would shorten cardiopulmonary bypass and procedural times and expedite post-operative recovery, due to lack of cooling and re-warming and decreased total body ischemia. Methods: Between 2012-2019, 65 infants underwent Norwood reconstruction at our institution. Those who underwent prior pulmonary artery banding were excluded, yielding a cohort of 51 infants who underwent primary Norwood reconstruction. Outcomes for patients who underwent Norwood reconstruction using STAR perfusion (n=21) were compared to those who underwent Norwood reconstruction with conventional techniques using regional cerebral perfusion only (n=30). STAR perfusion was performed with innominate artery cannulation as well as olive tip cannulas inserted into the opened descending aorta and native aortic root to provide continuous perfusion of the head, heart, and lower body throughout the procedure (Figure), under mild hypothermia (32-34°C). Results: Norwood reconstruction with STAR perfusion was associated with shorter median CPB times vs. conventional perfusion techniques (161 vs. 226 mins,p Conclusions: Norwood reconstruction with STAR perfusion is a safe technique that eliminates ischemic time to the heart and lower body and allows for the procedure to be performed at warmer temperatures. We found evidence of reduced CPB and operative times, as well as improved hemostasis, end-organ function, and post-operative recovery with this technique.

Background: Capillary refill is an early reflection of decreased end organ perfusion resulting in shunt of blood from the extremities to vital organs. Physicians rate this as the most important factor to guide therapeutic resuscitation efforts. However, literature shows that providers have significant variability in their bedside measurement of capillary refill. Objective: The objective of this study was to develop and evaluate a novel technology to detect capillary refill in a noninvasive and rapid manner through an iterative, problem-based innovation approach. Methods: This was a prospective study at a level 1 tertiary care hospital of patients presenting to an emergency department. The research team used in a new technology that utilizes light transmission and pressure to objectively quantify extremity capillary refill time. This measurement was compared to manual refill time performed by a trained study provider. Results: Through an iterative approach the team developed a platform technology and enrolled 63 subjects in this prospective trial. 9 subjects had inadequate data and so the final cohort consisted of 54 subjects with both manual capillary refill time and the new novel technology measurement. The device measured capillary refill time showed a high degree of correlation to manual estimate of capillary refill time with a Pearson coefficient of 0.7. Conclusions: Novel technology to measure capillary refill time can significantly improve the treatment and care of multiple medical conditions that rely on timely diagnosis and initiation of resuscitation in the emergency department and other care settings. This study shows a modest correlation of technology with automated signal processing algorithms for noninvasive measurement of capillary refill. Integration of this technology into standard hospital monitors has broad applicability to make capillary refill a standard vital sign.

Introduction: Current screening modalities for peripheral artery disease (PAD) lack sensitivity especially in the elderly and diabetics, and there is an unmet need for early detection of restenosis after revascularization. Hypothesis: We have hypothesized that arterial stenosis is associated with adaptive arteriolar vasodilatation, which alters the downstream perfusion dynamics and prolongs the initial phase of the perfusion upstroke. These changes can be utilized for quantification of the arterial stenosis severity. Methods: We measured the lower leg perfusion with impedance plethysmography and compared it with other modalities used in the clinic and the gold-standard angiography. The various phases of the perfusion wave were identified by analyzing the first and second derivatives of the plethysmography. The signals were acquired from PAD patients before and after they underwent revascularization, to validate the ability to detect stenosis and successful revascularization. Results: Eighteen consenting patients were recruited (61±10 years old) and nineteen legs were treated. The perfusion upstroke encompasses 2 or 3 distinctive phases. An initial slow phase that is followed by a brisk upstroke and a final sallower augmentation in some patients. The slow phase duration (SPd) was 113±45 ms in extremities with above-knee (AK) arterial stenosis (n=17) while significantly shorter SPd of 26±0 ms was observed in limbs without AK stenoses (P = 0.011). In the AK extremities, the SPd significantly decreased to 52±40 ms after successful revascularization (P Conclusions: Analysis of the perfusion dynamic provides a gamut of precious indices. The SPd is a novel index that can detect and quantify the severity of arterial stenosis. The technology can significantly improve the surveillance of PAD patients and may be used for early detection of restenosis.

Introduction: We aimed to clarify clinical implications of intrarenal hemodynamics (congestion and hypoperfusion) assessed by intrarenal Doppler ultrasonography (IRD) and their prognostic impacts in patients with heart failure (HF). Methods and Results: We performed IRD and measured interlobar renal artery velocity time integral (VTI) and intrarenal venous flow (IRVF) patterns (monophasic or non-monophasic pattern) to assess intrarenal hypoperfusion and congestion in HF patients (n=341). These patients were categorized based on 1) VTI: high VTI (VTI ≥ 14.0 cm, n=231) or low VTI (VTI < 14.0 cm, n=103); and 2) IRVF: monophasic (n=36) or non-monophasic (n=305) pattern. We performed right-heart catheterization, and examined post-discharge cardiac event rate such as cardiac death and rehospitalization due to worsening HF. Regarding renal perfusion, cardiac index was positively correlated with VTI (R=0.270, P=0.040). Concerning renal congestion, levels of right atrial pressure were higher in monophasic pattern than in non-monophasic pattern (9.0 vs. 7.2 mmHg, P=0.029). Importantly, HF patients with low VTI and a monophasic IRVF pattern (subset 4) had the highest cardiac event rate ( Figure ). In the Cox proportional hazard analysis, the combination of low VTI and a monophasic IRVF pattern was found to be a strong predictor of cardiac events (HR 8.357, 95% CI 3.365-20.752). Conclusion: Intrarenal hypoperfusion and congestion assessed by IRD imaging reflected cardiac output and right atrial pressure, and was useful to risk-stratify HF patients.

Introduction: Myocardial perfusion is enhanced during periods of elevated cardiomyocyte oxygen consumption. The coupling between blood flow and myocardial oxygen demand is mediated by redox-dependent signals, yet the influence of cardiac workload on arterial myocyte pyridine nucleotide redox status is unknown. Hypothesis: Increased cardiac workload acutely alters intramyocardial arterial myocyte [NADH] i :[NAD + ] i . Methods and Results: The genetically-encoded fluorescent biosensor Peredox-mCherry was used to monitor real-time changes in intracellular [NADH] i :[NAD] i in isolated arterial myocytes. Treatment of cells expressing Peredox-mCherry with a range of [lactate] o :[pyruvate] o resulted in significant changes in green (Ex,Em: 400, 510 nm): red (Ex,Em: 585,615 nm) fluorescence ratio (1.903 ± 0.010, 10 mM lactate, 1.072 ± 0.073, 10 mM pyruvate), affording accurate quantification of arterial myocyte [NADH] i :[NAD] i . Increased arterial myocyte [NADH] i :[NAD] i was observed in response to reductions in oxygen tension from 5% (0.0021 ± 0.0001) to 1% (0.0050 ± 0.0001; Pi :[NAD] i was not altered by direct stimulation of arterial myocytes with the beta-adrenergic agonist isoproterenol (1 μM; P = .0630). Furthermore, biosensor-expressing arterial myocytes in co-culture with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) recapitulated the effects of elevated [lactate] o :[pyruvate] o and reduced oxygen tension, exhibiting frequency-dependent increases in [NADH] i :[NAD] I following electrical pacing (e.g., ~3.3 fold increase in 3 Hz vs. unpaced; Pi :[NAD] i , compared with those in hearts treated with vehicle (3.17 ± 0.23 vs. 2.21 ± 0.11; P Conclusions: Our results indicate that acute increases in cardiac workload promote the elevation of [NADH] i :[NAD] i in intramyocardial arterial myocytes.

Introduction: Implantation of adipose-derived regenerative cells (ADRC) is a promising novel strategy to augment angiogenesis and blood perfusion recovery in ischemic diseases with no other therapeutic option. However, there is a clinical concern underlying therapeutic angiogenesis that implantation of ADRC may promote tumor growth and metastasis via remote angiogenesis. Accordingly, we tested whether therapeutic angiogenesis with ADRC against hindlimb ischemia (HLI) would affect remote tumor growth and angiogenesis in a tumor-bearing mouse ischemic hindlimb model. Methods and results: B16F10-Luc (murine melanoma cells expressing luciferase, 1x106 cells/animal) were implanted to C57BL/6J mice’s (male, 8-10 weeks old, n=10) back. Mice were subjected to unilateral HLI surgery one day after tumor implantation. Then, mice were randomly assigned to the control group or the ADRC group (n=5 for each). ADRC (1x106 cells/animal) or PBS were implanted/injected into ischemic hindlimb muscles one day after the surgery. Blood perfusion recovery in HLI by laser Doppler perfusion imaging system and tumor size by a caliper were measured every week up to 21 days after surgery. At POD 21, tumor weight and luciferase activity in primary tumors obtained by in vivo bioluminescence imaging system were also evaluated. Immunohistochemistry by CD31 or LYVE1 staining was performed to detect feeder arteries or outflow lymphatic vessels in tumors. The results demonstrated that better blood perfusion recovery and more capillary density in HLI was observed in the ADRC group than in the control group (p Conclusions: Our data indicated that therapeutic angiogenesis with ADRC implantation against HLI did not promote remote tumor growth, angiogenesis and metastasis.

Introduction: Screening for coronary artery disease in patients being considered for kidney transplant is common to stratify morbidity and mortality risk, but the optimal strategy, and its impact on outcomes, remains unclear. Here we test the hypothesis that myocardial perfusion imaging (MPI) abnormalities, left ventricular ejection fraction (LVEF), or coronary artery calcium (CAC) score are associated with all-cause mortality in potential kidney transplant recipients at Emory University Hospital (EUH). Methods: In a retrospective chart review, we assessed the relationship between patient demographics, single-photon emission MPI results, and CAC scoring with post-evaluation outcomes at 5 years in consecutive patients referred for pre-transplant stress testing at EUH in 2015. Mann-Whitney U and Chi-Square tests assessed between-group differences in continuous and categorical variables, respectively. Multivariate analysis was performed using logistic regression models. Results: During the study period, 589 patients (mean age 54 years; SEM 0.512, 58% male, 65% African American) underwent MPI and 424 also underwent CAC scoring. Overall, 90 patients (15%) had abnormal MPI (defined as any fixed or reversible defect) and 54 (9%) died during follow up. Age (mean 53.2 years; SEM 0.533 vs. 57.7 years; SEM 1.73, p=0.008), previous coronary artery bypass graft (CABG) (2.06% vs. 7.41%, p=0.017), and myocardial infarction (MI) post-evaluation (4.11% vs. 18.5%, p Conclusions: Age and MI post-evaluation are associated with increased mortality in potential kidney transplant recipients referred for stress testing at EUH. We found no association between MPI abnormalities, LVEF, or CAC score and all-cause mortality.

Introduction: Myocardial perfusion pressure (MPP), defined as the aortic diastolic pressure minus the central venous diastolic pressure, is an important determinant of return of spontaneous circulation (ROSC) in cardiac arrest. However, measuring MPP requires both arterial and venous catheters and is often not possible. When only invasive arterial monitoring is available, diastolic blood pressure (DBP) is suggested as a surrogate for MPP during resuscitation and is also associated with survival after cardiac arrest. Hypothesis: We hypothesized that DBP measured during chest compression delivery would mirror MPP during resuscitation and both would be associated with survival. Methods: We performed a retrospective, secondary analysis of 102 swine resuscitations. Pediatric swine underwent asphyxial cardiac arrest and were resuscitated with predefined periods of basic and advanced life support (BLS and ALS). MPP and DBP were recorded every 30 s during chest compression delivery. Results: For both survivors and non-survivors, DBP mirrored MPP throughout resuscitation ( Figure 1A, B ). During BLS, both MPP and DBP were significantly greater in survivors (MPP: 8.5 0.6 vs. 1.1 0.5 mmHg; p < 0.0001; DBP 17.3 0.6 vs. 8.7 0.5 mmHg; p < 0.0001). During ALS, MPP and DBP were greater in survivors than non-survivors (MPP: 20.5 1.0 vs. 0.7 0.3 mmHg; p < 0.0001; DBP 32.3 0.9 vs. 8.8 0.2 mmHg; p < 0.0001). During ALS, the magnitude of change in both MPP and DBP after the first epinephrine administration in survivors was greater than in non-survivors (MPP: 24.4 3.3 vs. 4.8 0.9 mmHg; p < 0.0001; DBP: 24.5 3.1 vs. 5.4 0.8 mmHg; p < 0.0001). Conclusion: These observations confirm that both DBP and MPP are associated with survival in cardiac arrest and validate the use of DBP as a surrogate for MPP. Figure 1: Myocardial perfusion pressure (MPP) and diastolic blood pressure (DBP) during BLS and ALS in survivors (A) and non-survivors (B). Arrows indicate administration of epinephrine during ALS.

Introduction: Because most studies have excluded arrhythmic patients, the prognostic value of stress perfusion CMR in patients with atrial fibrillation (AF) is unknown. Hypothesis: To assess the feasibility and the prognostic value of vasodilator stress perfusion CMR in patients with AF. Methods: Between 2008 and 2018, consecutive patients with suspected or stable chronic coronary artery disease (CAD) and AF referred for vasodilator stress perfusion CMR were included and followed for the occurrence of major adverse cardiovascular events (MACE), defined by cardiovascular death or non-fatal myocardial infarction. The diagnosis of AF was defined by 12-lead electrocardiogram before and after CMR. Univariable and multivariable Cox regressions were performed to determine the prognostic value of inducible ischemia or late gadolinium enhancement (LGE) by CMR. Results: Of 639 patients (72±9 years, 77% men), 602 (94%) completed the CMR protocol, and 538 (89%) completed the follow-up (median 5.1 years); 80 had a MACE. Stress CMR was well tolerated. Using Kaplan-Meier analysis, the presence of ischemia or LGE was associated with the occurrence of MACE (HR 7.56; 95% CI: 4.86-11.80 and HR 2.41; 95% CI: 1.55-3.74 respectively; for both p p Conclusions: In patients with AF, stress perfusion CMR is feasible, safe and has a good discriminative prognostic value to predict the occurrence of MACE.

Introduction: Several studies have demonstrated the consistently high prognostic value of stress perfusion cardiovascular magnetic resonance (CMR). This prognostic value in patients with known myocardial infarction (MI) is poorly described. Hypothesis: To assess the prognostic value of vasodilator stress perfusion CMR in patients with known MI. Methods: We prospectively included consecutive patients with known MI referred for vasodilator stress CMR with dipyridamole. They were followed for the occurrence of major adverse cardiovascular events (MACE) defined as cardiac death or recurrent non-fatal myocardial infarction (MI). Results: Of 1602 patients with known MI (68 ± 17 years, 78% men), 1556 (97%) completed the CMR protocol, and among those 1401 (90%) completed the follow-up (median follow-up 5.7 (3.9-7.6) years). Reasons for failure to complete CMR included ECG-gating problems (n=13), intolerance to stress agent (n=12), renal failure (n=12), declining participation (n=4) and claustrophobia (n=5). Stress CMR was well tolerated without occurrence of death or severe adverse event. Patients without inducible ischemia experienced a substantially lower annual event rate of MACE (3.1%) than those with 1 or 2 segments of ischemia (4.5%), than those with 3 to 5 segments of ischemia (21.5%), than those with 6 or more segments of ischemia (45.7%, for all pp p Conclusions: Stress CMR is technically feasible and has a good discriminative prognostic value to predict the occurrence of MACE in patients with known MI.

Introduction: Our clinical experience in cardiac surgery teaches us that a subset of patients show signs of unexplained end-organ hypoperfusion after an otherwise unremarkable procedure. The objective of this study is to comprehensively examine cardiopulmonary bypass (CPB) management parameters and end-organ perfusion. Hypothesis: Despite advancements in CPB management, CPB does not provide adequate end-organ perfusion. Methods: Retrospective analysis was performed using data from 1462 cardiac operations performed from 2/2019 to 4/2020, excluding circulatory arrest and non-CPB cases. CPB management parameters included cardiac index (CI), mean arterial pressure (MAP), vasopressor-phenylephrine use, lines and bladder temperatures, hemoglobin (Hgb), indexed oxygen delivery (iDO 2 ), and intravenous fluid infusion. End-organ perfusion markers included lactate levels, mixed venous oxygen saturation (SvO 2 ), oxygen consumption (VO 2 ), and arterial pH. Results were expressed as means with first standard deviation. Results: During CPB, CI, MAP, iDO 2 and Hgb were 2.2 ± 0.4 l/min/m 2 , 67 ± 16 mmHg, 270 ± 71 ml/min/m 2 , and 9.3 ± 1.6 g/dl respectively (Figure 1). Phenylephrine cumulative dose increased with CPB duration, 11 ± 9 mg at 1h and 20 ± 15 mg at 2h. Fluid administration showed similar trend. Core mean temperature was maintained with nadir being 33.5 ± 3.4°C. SvO 2 rapidly increased with institution of CPB, peaking at 85%, and then slowly declined. Inversely, VO 2 dropped and then slowly increased. Lactate continuously increased with duration of CPB. Conclusions: Our findings suggest that despite hemodynamic values being maintained at goal by continuously high vasopressor use and fluid administration during CPB, tissue perfusion was inadequate.Our study suggests that, using data-driven approaches, it may be time to revisit the assumptions regarding all aspects of managing CPB.

Introduction: Peripheral vascular disease (PVD) is a major cause of morbidity in the US. Vascular endothelial growth factor (VEGF) induces angiogenesis through Fetal-Liver Kinase-1 (Flk-1 or VEGFR-2), an endothelial tyrosine kinase receptor which activates a downstream signaling cascade involving MAPKAPKinase2 (MK2). We have demonstrated poorer recovery in hind limb ischemia models using Flk-1 +/- and MK2 -/- knockout mice compared to wild type mice. We also demonstrated overespression of Pellino1 (Peli1), an E3 Ligase, can attenuate ischemic damage and increase neovascularization and survivability of ischemic myocardium and skin flaps. Therefore, in this study we aim to show if Peli1 gene therapy can help rescue function in Flk-1 +/- and MK2 -/- after femoral artery ligation. Methods: 8-12 week-old Flk-1 +/- and MK2 -/- mice were subjected to femoral artery ligation of the right hind limb and treated with either adenovirus carrying Peli1 (Ad.Peli1) or LacZ (Ad.LacZ), for a total of 4 groups: Flk-1 +/- Ad.LacZ, Flk-1 +/- Ad.Peli1, MK2 -/- Ad.LacZ and MK2 -/- Ad.Peli1. Perfusion was assessed with laser Doppler imaging preop and on days 0, 3, 7, 14, 21 and 28. Extent of limb ischemia was also quantified using a standardized scoring scale (0 = no ischemia, to 8 = full limb amputation). Results: Perfusion was significantly higher in the Flk-1 +/- Ad.Peli1 group compared to Flk-1 +/- Ad.LacZ on days 7 (0.19±0.03 vs 0.08±0.02), 14 (0.41±0.07 vs 0.09±0.03), 21 (0.33±0.05 vs 0.14±0.05) and 28 (0.31±0.05 vs 0.15±0.02) (n=9-15, p+/- Ad.Peli1 group when compared to Flk-1 +/- Ad.LacZ on days 3 (1.89±0.56 vs 4.88±0.85), 7 (2.11±0.54 vs 5.69±0.92), 14 (2.18±0.52 vs 6.31±1.02), 21 (1.83±0.58 vs 6.50±1.05) and 28 (1.67±0.62 vs 6.50±1.05) (n=8-9, p-/- Ad.Peli1 and MK2 -/- Ad.LacZ groups. Values are mean +/- SEM. Conclusions: Treatment with Ad.Peli1 resulted in improved perfusion and decreased tissue ischemia in Flk-1 +/- mice. These results were not paralleled in MK2 -/- mice suggesting that Peli1 acts upstream to MK2 but downstream of VEGF/Flk-1. Overall, Peli1 gene therapy is a promising candidate in the management of PVD.

Introduction: Cardiovascular magnetic resonance imaging (CMR) has emerged as an accurate technique that can assess ventricular function, stress myocardial perfusion, and viability, without radiation. Recent studies have shown that stress CMR would be the best test to predict obstructive coronary artery disease (CAD) with a good safety. Hypothesis: To assess the feasibility and incidence of immediate complications of stress CMR in a tertiary Cardiovascular Center with CMR Laboratory dedicated. Methods: Prospective registry of vasodilator stress CMR in a French center with CMR expertise included all consecutive patients referred for vasodilator stress perfusion CMR to detect an obstructive CAD between 2008 and 2020. Stress CMR was performed at 1.5 T using dipyridamole. The clinical and demographic data, quality of test, CMR findings, haemodynamic data, and complications were prospectively recorded. Results: Stress CMR was performed in 35,157 patients (98.2% of requested). The study could not be performed due to claustrophobia in 0.3%. Quality was optimal in 93.1%, suboptimal in 6.4%, and poor in 0.5% of studies. Images were diagnostic in 97.9% of patients. No patient died or had acute myocardial infarction during the test. Moreover, 56 patients (0.16%) had severe immediate complications, and one anaphylactic shock post-gadolinium. The only factor significantly associated with higher incidence of serious complications was the detection of inducible ischaemia (p Conclusions: Performance of stress CMR is safe with very high image rate of satisfactory quality to perform the diagnosis in a referral population. Inducible ischaemia was the only factor identified which was associated with serious complications.

Introduction: Compensatory angiogenic response to ischemia is often insufficient in maintaining adequate tissue perfusion resulting in critical limb ischemia and amputation. Identifying a novel mechanism by which angiogenesis occurs in these conditions is clinically relevant. We recently uncovered that an increase in 20-HETE, an arachidonic acid metabolite of CYP4A/F ω-hydroxylases, regulates post-ischemic angiogenesis. However, the underlying mechanism resulting in this increase is unknown. Hypothesis: Neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to post-ischemic 20-HETE increases that drive angiogenesis. Methods: Hindlimb ischemia was established in mice depleted of neutrophils, macrophages, and MPO (MPO -/- ). Angiogenesis was assessed by laser doppler perfusion imaging and micro-vessel density quantitation in the hindlimb gracilis muscles. MPO and HOCl were detected in these tissues using immunohistochemistry and a HOCl-specific fluorophore. We also determined the effects of MPO and HOCl on 20-HETE production, the expression of 20-HETE synthase CYP4A11, and hypoxia inducible factor-1α in cultured endothelial cells (EC) using LC/MS/MS, real time-PCR and western blot analysis, respectively. Results: We found that ischemia failed to increase 20-HETE production in mice depleted of neutrophils and MPO (13 ± 1.5 vs 35 ± 5 and ~2 ± .25 vs 35 ± 5 pg/mg of protein, respectively), accompanied with a decreased post-ischemic angiogenic phenotype. We also detected the formation of MPO and HOCl in post-ischemic gracilis muscles. MPO and HOCl also significantly stimulate CYP4A11 expression and 20-HETE production (40±12 vs 8±5 pg/mg of protein) in EC. Furthermore, HOCl quickly induces CYP4A11 mRNA/protein expression (2-fold,) and the protein expression of HIF-1α (2-fold) in as little as 15 min. Conclusion: Our studies establish for the first time that neutrophil-derived MPO and HOCl are responsible for promoting 20-HETE increases that critically drive angiogenesis post ischemia. Thus, identifying these novel mediators can further future therapeutic strategies to balance angiogenic responses during ischemia as well as treating diseases that are associated with abnormal angiogenesis.

Supplemental Digital Content is available in the text., Background: Myocardial perfusion reflects the macro- and microvascular coronary circulation. Recent quantitation developments using cardiovascular magnetic resonance perfusion permit automated measurement clinically. We explored the prognostic significance of stress myocardial blood flow (MBF) and myocardial perfusion reserve (MPR, the ratio of stress to rest MBF). Methods: A 2-center study of patients with both suspected and known coronary artery disease referred clinically for perfusion assessment. Image analysis was performed automatically using a novel artificial intelligence approach deriving global and regional stress and rest MBF and MPR. Cox proportional hazard models adjusting for comorbidities and cardiovascular magnetic resonance parameters sought associations of stress MBF and MPR with death and major adverse cardiovascular events (MACE), including myocardial infarction, stroke, heart failure hospitalization, late (>90 day) revascularization, and death. Results: A total of 1049 patients were included with a median follow-up of 605 (interquartile range, 464–814) days. There were 42 (4.0%) deaths and 188 MACE in 174 (16.6%) patients. Stress MBF and MPR were independently associated with both death and MACE. For each 1 mL·g-1·min-1 decrease in stress MBF, the adjusted hazard ratios for death and MACE were 1.93 (95% CI, 1.08–3.48, P=0.028) and 2.14 (95% CI, 1.58–2.90, P

Background:Myocardial perfusion reflects the macro- and microvascular coronary circulation. Recent quantitation developments using cardiovascular magnetic resonance (CMR) perfusion permit automated measurement clinically. We explored the prognostic significance of stress myocardial blood flow (MBF) and myocardial perfusion reserve (MPR, the ratio of stress to rest MBF).Methods:A two center study of patients with both suspected and known coronary artery disease referred clinically for perfusion assessment. Image analysis was performed automatically using a novel artificial intelligence approach deriving global and regional stress and rest MBF and MPR. Cox proportional hazard models adjusting for co-morbidities and CMR parameters sought associations of stress MBF and MPR with death and major adverse cardiovascular events (MACE), including myocardial infarction, stroke, heart failure hospitalization, late (>90 day) revascularization and death.Results:1049 patients were included with median follow-up 605 (interquartile range 464-814) days. There were 42 (4.0%) deaths and 188 MACE in 174 (16.6%) patients. Stress MBF and MPR were independently associated with both death and MACE. For each 1ml/g/min decrease in stress MBF the adjusted hazard ratio (HR) for death and MACE were 1.93 (95% CI 1.08-3.48, P=0.028) and 2.14 (95% CI 1.58-2.90, PConclusions:In patients with known or suspected coronary artery disease, reduced MBF and MPR measured automatically inline using artificial intelligence quantification of CMR perfusion mapping provides a strong, independent predictor of adverse cardiovascular outcomes.

Introduction: Recent evidence has demonstrated that closed chest compressions (CC) directly over the left ventricle (LV) during traumatic cardiopulmonary arrest (TCPA) improve hemodynamics and return of spontaneous circulation (ROSC) when compared to traditional chest compressions. Selective Aortic Arch Perfusion (SAAP) is also able to improve hemodynamics as well as control hemorrhage during TCPA. Hypothesis: We hypothesized that LV compressions would increase ROSC when compared to traditional compressions in an animal model of TCPA where SAAP was being used. We also compared LV and Traditional compressions with SAAP to historical controls that received compressions without SAAP. Methods: Transthoracic echo was used to mark the location of the aortic root and the center of the left ventricle on animals (n=24, 12 in each arm) which were randomized to receive chest compressions in one of the two locations. One third of each animal’s blood volume was removed and ventricular fibrillation (VF) was induced to simulate traumatic cardiac arrest. After a period of ten minutes of VF, basic life support (BLS) with mechanical CPR was initiated and performed for ten minutes followed by advanced life support (ALS) and blood transfusion for an additional ten minutes. The SAAP balloon was inflated at minute 6 of BLS. Hemodynamic variables were averaged every two minutes. The same model was used for historical controls (n=26, 13 in each arm) without the use of SAAP, so comparisons were treated as a single study with a factorial design. Results: There were no differences in hemodynamic variables, ROSC (p=0.67) or survival (p=0.64) between SAAP groups. However, when the LV animals were compared to historic controls without SAAP, LV animals had higher ROSC (p=0.02) and survival rates (p=0.04) than Traditional animals. Conclusions: SAAP does not appear to show a synergistic effect with LV compressions, however, SAAP combined with LV compressions improves ROSC and survival when compared to Traditional compressions in our swine model of TCPA. More investigation into SAAP and compression position should be studied to improve treatment of TCPA.

Introduction: Derangement of the cerebrovascular unit (CVU) following ischemia-reperfusion from cardiac arrest contributes to overall brain injury. MRI can detect perfusion changes, but imaging timing and severity of injury vary in the clinical population. Hypothesis: We hypothesized that a large-animal model with fixed periods of global ischemia followed by standardized reperfusion and time to image acquisition would demonstrate a reliable gradient of perfusion changes. Such a model would allow neuroprotective strategies to be tested using early imaging biomarkers to quantify effects on cerebral perfusion. Methods: 10 healthy swine were placed under isoflurane anesthesia and underwent control MRI with dynamic susceptibility contrast (DSC) perfusion. Following imaging, animals were randomized to sham, 10, 15, 20, and 30-minute arrest groups, after which extracorporeal membrane oxygenation (ECMO) catheters were placed in the distal aorta and right atrium. Ventricular fibrillation was then induced by a bipolar pacing catheter for the fixed period of ischemia, after which animals were reperfused at 2.8-3.5 L/min with unblended oxygenated blood. After 4 minutes they were defibrillated. Over the next 2 hours they were weaned from pump and decannulated with epinephrine and fluid boluses given for hemodynamic support. Animals were then re-imaged within 2 hours post-resuscitation using the same pre-arrest protocol. DSC post-processing was performed and regions of interest were drawn in the bilateral frontal lobes to quantify perfusion parameters. Results: Baseline hemodynamic parameters, including BP, Heart Rate, ETCO2, and SpO2 were similar between animals. When comparing pre- to post-arrest MRI, there was a linear correlation of increasing Cerebral Blood Volume (CBV) with ischemic duration (r=0.69, p=0.028), and downward trend of Cerebral Blood Flow (CBF) with duration (r=-0.48, p=0.164). Conclusion: MRI DSC perfusion abnormalities were observed early in the post arrest period following fixed periods of ischemia with CBV correlated with ischemia duration consistent with dysfunctional vasodilation and uncoupling of the CVU. CBF showed a downward trend with injury severity. The effect of vasopressors and fluids on these findings is unclear.

Introduction: Cardiac arrest (CA), an unexpected loss of appropriate electrical signaling in the heart, leads to a loss of blood circulation and decreased oxygen perfusion. Ischemia results in the generation of hydrogen peroxide and other reactive oxygen species (ROS), thereby causing damage to tissues. Currently, there are no available biomarkers to elucidate the severity of ischemic damage. Therefore, oxidation of the Amplex Red (AR) assay by ROS into its fluorescent product, resorufin, may be used as a marker to determine injury severity. Methods: Plasma isolated from human CA patients from North Shore University Hospital was obtained to determine ROS generation. A commercially available Amplex Red assay kit was used to measure the amount of resorufin produced after oxidation due to hydrogen peroxide, peroxynitrite, and other ROS. To verify our human findings, we arbitrarily assigned adult male Sprague-Dawley rats into three groups (control, 10 min cardiac arrest, and 20 min cardiac arrest) using our reliable asphyxia-induced cardiac arrest model. Results: Despite human variations, our data on human CA patients showed an increased amount of AR oxidation as a result of ischemia. Our 10 min CA rat experimental model verified that Amplex Red is capable of detecting hydrogen peroxide and peroxynitrite formation after ischemia. Rats with 20 mins of ischemia time also produced resorufin, confirming that ischemia induces AR oxidation. Removing horseradish peroxidase and adding catalase controls for hydrogen peroxide and peroxynitrite, which should decrease AR oxidation; however, we observed an increase in AR oxidation. Therefore, we added phenylmethyl sulfonyl acid (PMSF), an inhibitor of carboxylesterase, an enzyme also capable of oxidizing Amplex Red, which resulted in decreased AR oxidation. Conclusion: By accounting for peroxide and peroxynitrite species, the increase in Amplex Red oxidation in the plasma of cardiac arrest human patients and rats can be attributed to carboxylesterase activity. Our data corroborates the various mechanisms of AR oxidation in the setting of ischemia-reperfusion allowing the Amplex Red assay to be utilized as a potential tool for assessing the degree of ischemic damage resulting from cardiac arrest.

Introduction: Hypoxic ischemic brain injury (HIBI) results from loss of oxygen or perfusion during cardiac arrest and is the most common cause of death/disability in patients surviving to admission. Diagnostic tests are crucial to identify irreversible injury thresholds for neuroprognostication. MRI DTI can detect cellular injury in response to HIBI, but imaging timing and severity of hypoxia vary widely. Hypothesis: We hypothesized that a model with fixed periods of ischemia followed by both standardized reperfusion and time to image acquisition would demonstrate a reliable gradient of injury. Such a model would allow neuroprotective strategies to be tested using early imaging biomarkers to quantify effects on cytoarchitecture. Methods: Eleven healthy swine (49 + 5 kg) under isoflurane anesthesia underwent control MRI with DTI at b2000/20 directions. Two animals each were then randomized to sham, 10, 15, 20, and one to 30-minutes of arrest. For reperfusion, extracorporeal membrane oxygenation (ECMO) catheters were placed in the distal aorta and right atrium, after which ventricular fibrillation was induced by a bipolar pacing catheter. Following the fixed period of ischemia, animals were reperfused at 2.8-3.5 L/min with unblended oxygenated blood. After 4 minutes they were defibrillated. Over two hours they were weaned from pump and decannulated with epinephrine and fluid boluses for hemodynamic support. Animals were re-imaged within two hours post-resuscitation using the same pre-arrest protocol. Whole brain ADC measurements were performed in the gray and white matter. Results: Baseline hemodynamic parameters: BP, Heart Rate, End-Tidal CO2, and SpO2 were similar between animals. When comparing pre- to post-arrest MRI, there was a strong linear correlation between percent gray matter ADC change (r=-0.87, p Conclusion: MRI DTI changes observed following fixed periods of ischemia with ADC in gray and white matter correlated well with ischemia duration in the first few hours post arrest. These findings support the feasibility of MRI for measuring severity of insult in HIBI and an animal model that might serve as translational endpoint for neuroprotectant strategies.

Introduction: Cardiogenic shock (CS) is a condition of insufficient tissue perfusion due to cardiac dysfunction. Despite the improvement in prognosis due to aggressive reperfusion strategies, in-hospital mortality from CS is around 50%. Intra-aortic balloon pump (IABP) has been used for the management of CS. Percutaneous Ventricular Assist Devices (pVAD) such as the tandem heart and the Impella have been developed to treat CS. We carry out a meta-analysis comparing the IABP versus pVAD during CS. Method: Using PubMed, Cochrane Library, and EMBASE we searched for randomized clinical trials (RCTs) that compared pVAD versus IABP in patients CS. Result: We demonstrated no difference in short-term 30 days all-cause mortality (risk ratio [RR] 1.14 [CI: 0.70+/- 1.86]. The RR of acute kidney injury (AKI) for pVAD compared to IABP is 0.96 [CI: 0.46 +/- 2.00], for limb ischemia RR is 3.31 [CI: 1.04 +/- 10.51], for infection/sepsis 0.59 [CI: 0.19+/-1.80], for bleeding 2.41 [CI: 1.41+/- 4.13] and for vascular injury 3.75 [CI: 0.19 +/- 72.49]. Although pVAD provides superior hemodynamic support in patients with CS compared with IABP, the use of these more powerful devices did not improve early survival. The use of pVAD is associated with more adverse events compared with IABP. Discussion: The use of more vigorous hemodynamic support for CS does not associate with a 30 days survival benefit but could be associated with more adverse events. The reason for these findings is unclear but could be explained by the fact that decreased cardiac output seen in CS is only a single component of the multifaceted syndrome that involves several other molecular and hemodynamic mechanisms or merely because of the increase in the cardiac output by the IABP or the pVAD is inadequate to improve outcomes. Conclusion: Use of pVAD resulted in a better hemodynamic profile compared with IABP. However, this did not translate into improved 30-day survival. Also, patients treated with a pVAD have a higher risk of leg ischemia, AKI and, device-related bleeding.

Background: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1 , that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. Methods: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2–associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. Results: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6– Lsq1-3 ). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein– (n=29) expressing animals. BAG3 Ile81 , but not BAG3 Met81 , improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus–BAG3 Ile81 (n=9), but not BAG3 Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3 Met81 , BAG3 Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Conclusions: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Article, see p 1509 Heart failure (HF) remains one of the largest health problems in the Western world that caused an estimated 37.6 million HF or HF-related hospitalizations in the United States between 2001 and 2009.1 After an insult to the myocardium, cardiac output drops, which will lead to impairment of the systemic circulation and ultimately signs and symptoms of HF. Indeed, tissue and organ perfusion generally decreases in HF, despite the fact that local compensatory mechanisms are activated. To maintain sufficient perfusion of vital organs and peripheral tissues, vasoconstrictor mechanisms in the systemic arterial bed become active in response to stimulation of the sympathetic nervous and renin-angiotensin system.2 This has paved the way for the use of arterial vasodilators in HF because long-term systemic vasoconstriction leads to left ventricular hypertrophy and failure and ultimately refractory HF. As a consequence of left-sided HF, pulmonary congestion may occur. Previously, pulmonary congestion was considered mainly a marker of severity of left-sided HF, but there is increasing evidence that pulmonary congestion may also be actively contributing to the HF syndrome and impair prognosis.3 Along with previous attempts to indirectly monitor signs related to pulmonary congestion in HF,4 there is a (renewed) interest in more directly measuring pulmonary artery (PA) pressures in HF. Moreover, elevated PA pressures have been shown to strongly associate with an adverse prognosis and increased risk for hospitalization in patients with HF (Figure).5 For this reason, the pulmonary circulation as a therapeutic target in HF has gained increasing interest in recent years. Figure. Association between pulmonary pressures and outcomes in outpatients with heart failure …

Background: Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Methods: Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 8 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. Results: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. Conclusions: Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. Clinical Trial Registration: URL: http://clinicaltrials.gov . Unique identifier: NCT01566890.