Lifetime evaluation of left ventricular structure and function in male ApoE null mice after gamma and space-type radiation exposure.

The space radiation (IR) environment contains high charge and energy (HZE) nuclei emitted from galactic cosmic rays with the ability to overcome current shielding strategies, posing increased IR-induced cardiovascular disease risks for astronauts on prolonged space missions. Little is known about the effect of 5-ion simplified galactic cosmic ray simulation (simGCRsim) exposure on left ventricular (LV) function. Three-month-old, age-matched male Apolipoprotein E (ApoE) null mice were irradiated with 137Cs gamma (γ; 100, 200, and 400 cGy) and simGCRsim (50, 100, 150 cGy all at 500 MeV/nucleon (n)). LV function was assessed using transthoracic echocardiography at early/acute (14 and 28 days) and late/degenerative (365, 440, and 660 days) times post-irradiation. As early as 14 and 28-days post IR, LV systolic function was reduced in both IR groups across all doses. At 14 days post-IR, 150 cGy simGCRsim-IR mice had decreased diastolic wall strain (DWS), suggesting increased myocardial stiffness. This was also observed later in 100 cGy γ-IR mice at 28 days. At later stages, a significant decrease in LV systolic function was observed in the 400 cGy γ-IR mice. Otherwise, there was no difference in the LV systolic function or structure at the remaining time points across the IR groups. We evaluated the expression of genes involved in hemodynamic stress, cardiac remodeling, inflammation, and calcium handling in LVs harvested 28 days post-IR. At 28 days post-IR, there is increased expression of Bnp and Ncx in both IR groups at the lowest doses, suggesting impaired function contributes to hemodynamic stress and altered calcium handling. The expression of Gals3 and ß-Mhc were increased in simGCRsim and γ-IR mice respectively, suggesting there may be IR-specific cardiac remodeling. IR groups were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). No lower threshold was determined using the observed dose-response curves. These findings do not exclude the possibility of the existence of a lower IR threshold or the presence of IR-induced cardiovascular disease (CVD) when combined with additional space travel stressors, e.g., microgravity.

The Relationship Between Serum Angiotensin Converting Enzyme Level and the Decision to Escalate Treatment of Sarcoidosis.

PURPOSE: We performed a retrospective analysis of a sarcoidosis cohort who had sACE obtained at their initial clinic visit, but the treating physician was blinded to the results. We examined the relationship between sACE and the treating physician's decision to escalate sarcoidosis treatment. METHODS: Treatment was considered escalated if the prednisone dose was increased or if the prednisone dose was not changed but an additional anti-sarcoidosis drug was added or the dose was increased. RESULTS: 561 sarcoidosis patients were analyzed. The most common target organ was the lung (84%). Using a cut-off of > 82 units/L for an elevated sACE, 31/82 (38%) with an elevated sACE had treatment escalation whereas 91/497 (18%) had treatment escalation with a normal sACE (p < 0.0001). For the need of treatment escalation, a sACE (cut-off of > 82) had sensitivity 0.25, specificity 0.89, positive predictive value 0.38, negative predictive value 0.81. These results were not appreciably different using other sACE cut-off values such as 70, 80, 90, or 100. A multivariable logistic regression model that included demographics, the target organ, spirometry results estimated that sACE level and lower FVC were significantly associated with the likelihood of treatment escalation. These findings held when sACE > 82 replaced sACE level in the multivariable logistic regression model. CONCLUSIONS: Although there was a strong correlation between sACE at the initial sarcoidosis clinic visit and subsequent treatment escalation of sarcoidosis, the predictive power was such that sACE is not adequately reliable to be used in isolation to make this determination.

Lifetime Evaluation of Left Ventricular Structure and Function in Male C57BL/6J Mice after Gamma and Space-Type Radiation Exposure.

The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with 137Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers Tgfß1, Mcp1, Mmp9, and ßmhc, suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100-200 cGy for γ-IR, and 50-100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity.

Outcomes of prednisone-tapering regimens for cardiac sarcoidosis: A retrospective analysis demonstrating a benefit of infliximab.

BACKGROUND: The optimal treatment strategy for cardiac sarcoidosis has not been standardized. We examined the effectiveness of three prednisone-tapering treatment regimens for cardiac sarcoidosis. METHODS: We retrospectively reviewed prednisone-tapering treatment regimens for cardiac sarcoidosis that contained prednisone alone (P), prednisone plus methotrexate (P-M), and prednisone plus infliximab containing regimens (P-I). We defined the success of each regimen as the ability to lower the daily prednisone dose to 7.5 mg or less for 6 or more months without developing an adverse cardiac event. We also examined the lowest effective daily prednisone dose achieved without developing an adverse cardiac event. RESULTS: We identified 61 treatment regimens in 33 cardiac sarcoidosis patients that were analyzed. The success rate of prednisone-tapering regimens was significantly different P: 8/30, 27%; P-M: 3/23, 13%; P-I: 6/8, 75%., p = 0.04. The lowest effective daily prednisone dose for the regimens was also significantly different: P: 14.1 ± 10.1 mg; P-M: 16.9 ± 9.4 mg; infliximab: 7.8 ± 4.9 mg, (p = 0.03); by both measures the success was greatest with the P-I regimen. CONCLUSIONS: For the treatment of cardiac sarcoidosis, prednisone-tapering regimens containing infliximab are superior to those containing prednisone alone or prednisone plus methotrexate in terms of reaching 7.5 mg/day of prednisone for more than 6 months and achieving the lowest effective prednisone.

Endobronchial Aerosolized AAV1.SERCA2a Gene Therapy in a Pulmonary Hypertension Pig Model: Addressing the Lung Delivery Bottleneck.

A disappointing number of new therapies for pulmonary hypertension (PH) have been successfully translated to the clinic. Adeno-associated viral (AAV) gene therapy has the potential to treat the underlying pathology of PH, but the challenge remains in efficient and safe delivery. The aims of this study were (1) to test the efficacy of endobronchial aerosolization delivery for AAV1-mediated sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) gene therapy in a PH pig model and (2) to identify the most efficient airway administration modality for in-lung gene therapy in PH. We hypothesized that delivery to the distal bronchi increases lung viral uptake and avoids virus loss in off-target compartments. In part 1 of the study, PH was induced in pigs by surgically banding the pulmonary veins. Two months postsurgery, 1 × 1013 viral genomes (vg) of AAV1.SERCA2a or saline was endobronchially aerosolized using a bronchoscope. Two months after aerosolization, high vg copies (vgc) were detected in the lungs, accompanied by functional and morphometrical amelioration of PH. In part 2 of the study, we directly compared the endobronchial aerosolization gene delivery to the intratracheal aerosolization in PH pigs. Endobronchial delivery demonstrated higher viral expression (6,719 ± 927 vs. 1,444 ± 402 vgc/100 ng DNA, p = 0.0017), suggesting this delivery modality is a promising method for clinical AAV gene therapy for PH.

Comorbidities, sequelae, blood biomarkers and their associated clinical outcomes in the Mount Sinai Health System COVID-19 patients.

With the continuing rise of SARS-CoV2 infection globally and the emergence of various waves in different countries, understanding characteristics of susceptibility to infection, clinical severity, and outcomes remain vital. In this retrospective study, data was extracted for 39,539 patients from the de-identified Mount Sinai Health System COVID-19 database. We assessed the risk of mortality based on the presence of comorbidities and organ-specific sequelae in 7,032 CoV2 positive (+) patients. Prevalence of cardiovascular and metabolic comorbidities was high among SARS-CoV2+ individuals. Diabetes, obesity, coronary artery disease, hypertension, atrial fibrillation, and heart failure all increased overall mortality risk, while asthma did not. Ethnicity modified the risk of mortality associated with these comorbidities. With regards to secondary complications in the setting of infection, individuals with acute kidney injury and acute myocardial injury showed an increase in mortality risk. Cerebral infarcts and acute venous thromboembolic events were not associated with increased risk of mortality. Biomarkers for cardiovascular injury, coagulation, and inflammation were compared between deceased and survived individuals. We found that cardiac and coagulation biomarkers were elevated and fell beyond normal range more often in deceased patients. Several, but not all, inflammatory markers evaluated were increased in deceased patients. In summary, we identified comorbidities and sequelae along with peripheral blood biomarkers that were associated with elevated clinical severity and poor outcomes in COVID-19 patients. Overall, these findings detail the granularity of previously reported factors which may impact susceptibility, clinical severity, and mortality during the course of COVID-19 disease.

Long-Term Effects of Very Low Dose Particle Radiation on Gene Expression in the Heart: Degenerative Disease Risks.

Compared to low doses of gamma irradiation (γ-IR), high-charge-and-energy (HZE) particle IR may have different biological response thresholds in cardiac tissue at lower doses, and these effects may be IR type and dose dependent. Three- to four-month-old female CB6F1/Hsd mice were exposed once to one of four different doses of the following types of radiation: γ-IR 137Cs (40-160 cGy, 0.662 MeV), 14Si-IR (4-32 cGy, 260 MeV/n), or 22Ti-IR (3-26 cGy, 1 GeV/n). At 16 months post-exposure, animals were sacrificed and hearts were harvested and archived as part of the NASA Space Radiation Tissue Sharing Forum. These heart tissue samples were used in our study for RNA isolation and microarray hybridization. Functional annotation of twofold up/down differentially expressed genes (DEGs) and bioinformatics analyses revealed the following: (i) there were no clear lower IR thresholds for HZE- or γ-IR; (ii) there were 12 common DEGs across all 3 IR types; (iii) these 12 overlapping genes predicted various degrees of cardiovascular, pulmonary, and metabolic diseases, cancer, and aging; and (iv) these 12 genes revealed an exclusive non-linear DEG pattern in 14Si- and 22Ti-IR-exposed hearts, whereas two-thirds of γ-IR-exposed hearts revealed a linear pattern of DEGs. Thus, our study may provide experimental evidence of excess relative risk (ERR) quantification of low/very low doses of full-body space-type IR-associated degenerative disease development.

Successful Transduction with AAV Vectors after Selective Depletion of Anti-AAV Antibodies by Immunoadsorption.

Gene therapy with adeno-associated virus (AAV)-based vectors shows great promise for the gene therapeutic treatment of a broad array of diseases. In fact, the treatment of genetic diseases with AAV vectors is currently the only in vivo gene therapy approach that is approved by the US Food and Drug Administration (FDA). Unfortunately, pre-existing antibodies against AAV severely limit the patient population that can potentially benefit from AAV gene therapy, especially if the vector is delivered by intravenous injection. Here, we demonstrate that we can selectively deplete anti-AAV antibodies by hemapheresis combined with AAV9 particles coupled to Sepharose beads. In rats that underwent hemapheresis and immunoadsorption, luciferase expression was dramatically increased in the hearts and fully restored in the livers of these rats. Importantly, our method can be readily adapted for the use in clinical AAV gene therapy.

Primary Effect of SERCA 2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction.

Background SERCA 2a gene transfer ( GT ) improves mechano-electrical function in animal models of nonischemic heart failure Whether SERCA 2a GT reverses pre-established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno-associated virus serotype 1. SERCA 2a GT following myocardial infarction ( MI ). Methods and Results Pigs developed mechanical dysfunction 1 month after anterior MI , at which point they received intracoronary adeno-associated virus serotype 1. SERCA 2a ( MI + SERCA 2a) or saline ( MI ) and were maintained for 2 months. Age-matched naive pigs served as controls (Control). In vivo ECG -and-hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential ( AP ) mapping in controls, MI , and MI + SERCA 2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R' waves were present in MI but not MI + SERCA 2a pigs relative to controls. SERCA 2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI + SERCA 2a or control pigs were prone to pacing-induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI + SERCA 2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI + SERCA 2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA 2a GT decreased phosphoactive CAMKII -delta levels by >50%, leading to improved excitability at fast rates. Conclusions SERCA 2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA 2a GT on myocardial excitability, independent of altered mechanical function.

Chronic Pulmonary Artery Embolization Models in Large Animals.

A wide range of approaches have been described to develop animal models of pulmonary vascular disease (PVD). Clinical heterogeneity in patients with pulmonary hypertension (PH) has prompted development of different techniques to create PH models in several animal species with the objective to recapitulate specific PH/PVD phenotypes. Chronic thromboembolic PH (CTEPH) is a clinically important phenotype of PH with a documented prevalence of 0.4-9.1% in patients with history of pulmonary embolism. A well-established large animal model of CTEPH is thus necessary for studying this disease in preclinical research. Different experimental protocols with inconsistent outcomes have been reported in the literature.We have focused on characterizing PH large animal models in a common framework; pulmonary hemodynamics, right ventricular (RV) function, and histological characterization of PVD. This research framework allows optimal evaluation of novel diagnostic tools, as well as new therapeutic strategies. The purpose of this protocol is to describe approaches to create experimental CTEPH models using recurrent pulmonary embolizations of dextran microspheres in swine. The key features of this experimental modeling approach are (1) nonsurgical, fully percutaneous techniques, (2) a minimum of four embolization procedures, with 1-2 month time period, (3) mild to moderate PH hemodynamics (mean PA pressure increase ~20-60%), (4) severe pulmonary vascular remodeling, (5) mild RV remodeling, and (6) a high reproducibility and low mortality (<10%).

Modeling Pulmonary Hypertension: A Pig Model of Postcapillary Pulmonary Hypertension.

Pulmonary hypertension (PH) is a pathophysiological condition defined as an increase in mean pulmonary arterial pressure ≥25 mmHg at rest assessed by right heart catheterization.Based on hemodynamic criteria, precapillary PH is characterized by a mean pulmonary capillary wedge pressure ≤15 mmHg as opposed to the postcapillary PH by >15 mmHg. Postcapillary PH is one of the most common forms of PH, often caused by left ventricular dysfunction and heart failure.In this chapter, we describe protocols for creating a large animal model of postcapillary PH. It is induced by open chest surgery (lateral thoracotomy) to band the pulmonary veins. The model is characterized by low mortality, relatively easy surgical procedure with well reproducible results, and pulmonary and cardiac remodeling at the structural, functional, and molecular levels. The presence of right ventricular (RV) remodeling is of significant importance since right heart failure is the main cause of death in patients suffering from PH. One of the advantages of the model described in this chapter is that both adaptive and maladaptive forms of RV remodeling can be observed during the progression of the disease. This can help understand the progressive pathophysiology of RV failure in humans. Besides the description of the model, a detailed guidance of the RV functional assessment in pigs for both invasive (heart catheterization) and noninvasive (echocardiography) approaches is provided.

Increased afterload following myocardial infarction promotes conduction-dependent arrhythmias that are unmasked by hypokalemia.

Although the pathophysiological significance of resistant hypertension in post-myocardial infarction (MI) patients is established, mechanisms by which increased afterload in that setting worsens outcome are unclear. With regards to sudden cardiac death, whether increased afterload alters the electrophysiological substrate following MI is unknown. We established a new large animal model of chronic post-MI remodeling with increased afterload which exhibits widespread deposition of fibrosis in remote areas from the anterior MI, mimicking the disease phenotype of patients with advanced ischemic heart disease. We identified the mode-of-initiation and mechanism of arrhythmias which were consistently unmasked by hypokalemia in this clinically-relevant model.

In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models.

OBJECTIVES: The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaque-targeting abilities. BACKGROUND: HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS: Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 ((89)Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS: We observed distinct pharmacokinetic profiles for the two (89)Zr-HDL nanoparticles. Both apo A-I- and phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days' post-injection for both (89)Zr-HDL nanoparticles. In the porcine model, increased accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel's location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS: (89)Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL's main components (i.e., phospholipids, apo A-I).

Intratracheal Gene Delivery of SERCA2a Ameliorates Chronic Post-Capillary Pulmonary Hypertension: A Large Animal Model.

BACKGROUND: Pulmonary hypertension (PH) is characterized by pulmonary arterial remodeling that results in increased pulmonary vascular resistance, right ventricular (RV) failure, and premature death. Down-regulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) in the pulmonary vasculature leads to perturbations in calcium ion (Ca(2+)) homeostasis and transition of pulmonary artery smooth muscle cells to a proliferative phenotype. OBJECTIVES: We assessed the feasibility of sustained pulmonary vascular SERCA2a gene expression using aerosolized delivery of adeno-associated virus type 1 (AAV1) in a large animal model of chronic PH and evaluated the efficacy of gene transfer regarding progression of pulmonary vascular and RV remodeling. METHODS: A model of chronic post-capillary PH was created in Yorkshire swine by partial pulmonary vein banding. Development of chronic PH was confirmed hemodynamically, and animals were randomized to intratracheal administration of aerosolized AAV1 carrying the human SERCA2a gene (n = 10, AAV1.SERCA2a group) or saline (n = 10). Therapeutic efficacy was evaluated 2 months after gene delivery. RESULTS: Transduction efficacy after intratracheal delivery of AAV1 was confirmed by ß-galactosidase detection in the distal pulmonary vasculature. Treatment with aerosolized AAV1.SERCA2a prevented disease progression as evaluated by mean pulmonary artery pressure, vascular resistance, and limited vascular remodeling quantified by histology. Therapeutic efficacy was supported further by the preservation of RV ejection fraction (p = 0.014) and improvement of the RV end-diastolic pressure-volume relationship in PH pigs treated with aerosolized AAV1.SERCA2a. CONCLUSIONS: Airway-based delivery of AAV vectors to the pulmonary arteries was feasible, efficient, and safe in a clinically relevant chronic PH model. Vascular SERCA2a overexpression resulted in beneficial effects on pulmonary arterial remodeling, with attendant improvements in pulmonary hemodynamics and RV performance, and might offer therapeutic benefit by modifying fundamental pathophysiology in pulmonary vascular diseases.

Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression.

Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 µg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 µg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 µg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.

Advances in gene therapy for heart failure.

Chronic heart failure is expected to increase its social and economic burden as a consequence of improved survival in patients with acute cardiac events. Cardiac gene therapy holds significant promise in heart failure treatment for patients with currently very limited or no treatment options. The introduction of adeno-associated virus (AAV) gene vector changed the paradigm of cardiac gene therapy, and now it is the primary vector of choice for chronic heart failure gene therapy in clinical and preclinical studies. Recently, there has been significant progress towards clinical translation in this field spearheaded by AAV-1 mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene therapy targeting chronic advanced heart failure patients. Meanwhile, several independent laboratories are reporting successful gene therapy approaches in clinically relevant large animal models of heart failure and some of these approaches are expected to enter clinical trials in the near future. This review will focus on gene therapy approaches targeting heart failure that is in clinical trials and those close to its initial clinical trial application.