ABSTRACT
AIMS: Vein grafts are used for many indications, including bypass graft surgery and arterio-venous fistula (AVF) formation. However, patency following vein grafting or AVF formation is suboptimal for various reasons, including thrombosis, neointimal hyperplasia and adverse remodeling. Recently, endothelial to mesenchymal transition (EndMT) was found to contribute to neointimal hyperplasia in mouse vein grafts. We aimed to evaluate the clinical potential of inhibiting EndMT, and developed the first dedicated preclinical model to study the efficacy of local EndMT inhibition immediately prior to AVF creation. METHODS AND RESULTS: We first undertook pilot studies to optimize the creation of a femoral AVF in pigs and verify that EndMT contributes to neointimal formation. We then developed a method to achieve local in vivo SMAD3 knockdown by dwelling a lentiviral construct containing SMAD3 shRNA in the femoral vein prior to AVF creation. Next, in Phase 1, 6 pigs were randomized to SMAD3 knockdown or control lentivirus to evaluate the effectiveness of SMAD3 knockdown and EndMT inhibition 8 days after AVF creation. In Phase 2, 16 pigs were randomized to SMAD3 knockdown or control lentivirus and were evaluated to assess longer-term effects on AVF diameter, patency and related measures at 30 days after AVF creation.In Phase 1, compared to controls, SMAD3 knockdown achieved a 75% reduction in the proportion of CD31+ endothelial cells co-expressing SMAD3 (p<0.001), and also a significant reduction in the extent of EndMT (p<0.05). In Phase 2, compared to controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56±1.66 versus 4.26±1.71mm, p<0.01) and a reduced degree of stenosis (p<0.01). Consistent with this, neointimal thickness was reduced in the SMAD3 knockdown group (0.88±0.51 versus 0.45±0.19mm, p<0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (p<0.05). CONCLUSIONS: EndMT inhibition in a preclinical AVF model by local SMAD3 knockdown using gene therapy led to reduced neointimal hyperplasia, increased endothelialization and a reduction in the degree of AVF stenosis. This provides important proof-of-concept to pursue this approach as a clinical strategy to improve the patency of AVFs and other vein grafts.
ABSTRACT
Acute myocardial infarction continues to account for a growing burden of heart failure worldwide. Despite existing therapies, new approaches for reducing the extent of damage and better managing heart failure progression are urgently needed. Preclinical large animal models are a critical step in the translation of scientific discoveries toward clinical trials and therapeutic application. In this chapter, we detail methods to induce swine models of myocardial infarction through catheter-mediated approaches involving either temporary (ischemia-reperfusion) or permanent (thrombus injection or embolic coil) occlusions. These techniques are relatively low in invasiveness, while infarct size with corresponding cardiac dysfunction can be controlled by adjusting the location of coronary occlusion. We also describe methods for cardiac angiography and echocardiography in pigs. This is the second edition of a previously published chapter with modifications.
Subject(s)
Disease Models, Animal , Myocardial Infarction , Myocardial Reperfusion Injury , Animals , Myocardial Infarction/therapy , Myocardial Infarction/pathology , Swine , Myocardial Reperfusion Injury/therapy , Myocardial Reperfusion Injury/etiology , Myocardial Reperfusion Injury/pathology , Echocardiography/methods , Coronary Angiography/methods , Embolism/etiology , Embolism/therapy , Embolism/pathologyABSTRACT
Coronary artery dissection (CAD) is the intimal tearing of the coronary arterial wall and can be iatrogenic, spontaneous, or traumatic in origin. CAD is a rare but challenging condition that can cause significant hemodynamic compromise. Management strategies for CAD, such as the use of mechanical circulatory support devices, are available in the clinical setting. However, the incidence, etiology, and optimal management of CAD are not well-defined, emphasizing the need for adequate animal models in preclinical studies. Large animal models provide the human-like conditions necessary for testing and development of potential treatment strategies. In this chapter, we describe a method for the creation of a CAD swine model.
Subject(s)
Aortic Dissection , Coronary Vessels , Disease Models, Animal , Vascular Diseases/congenital , Animals , Swine , Coronary Vessels/pathology , Humans , Coronary Vessel Anomalies , Vascular Diseases/etiology , Vascular Diseases/pathology , Vascular Diseases/therapy , Coronary Artery Disease/pathologyABSTRACT
Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Acute mechanical left ventricular (LV) support has been suggested to improve infarct tissue perfusion. However, its regulatory mechanism remains unclear. We investigated the physiological mechanisms in six Yorkshire pigs, which were subjected to 90-min balloon occlusion of the left anterior descending artery. During the acute reperfusion phase, LV support using an Impella heart pump was initiated. LV pressure, coronary flow and pressure of the infarct artery were simultaneously recorded to evaluate the impact of LV support on coronary physiology. Coronary wave intensity was calculated to understand the forces regulating coronary flow. Significant increases in coronary flow velocity and its area under the curve were found after mechanical LV support. Among the coronary flow-regulating factors, coronary pressure was increased mainly during the late diastolic phase with less pulsatility. Meanwhile, LV pressure was reduced throughout diastole resulting in significant and consistent elevation of coronary driving pressure. Interestingly, the duration of diastole was prolonged with LV support. In the wave intensity analysis, the duration between backward suction and pushing waves was extended, indicating that earlier myocardial relaxation and delayed contraction contributed to the extension of diastole. In conclusion, mechanical LV support increases infarct coronary flow by extending diastole and augmenting coronary driving pressure. These changes were mainly driven by reduced LV diastolic pressure, indicating that the key regulator of coronary flow under mechanical LV support is downstream of the coronary artery, rather than upstream. Our study highlights the importance of LV diastolic pressure in infarct coronary flow regulation. KEY POINTS: Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Although mechanical left ventricular (LV) support has been suggested to improve infarct coronary flow, its specific mechanism remains to be clarified. LV support reduced LV pressure, and elevated coronary pressure during the late diastolic phase, resulting in high coronary driving pressure. This study demonstrated for the first time that mechanical LV support extends diastolic phase, leading to increased infarct coronary flow. Future studies should evaluate the correlation between improved infarct coronary flow and resulting infarct size.
Subject(s)
Myocardial Infarction , Ventricular Function, Left , Animals , Swine , Diastole/physiology , Ventricular Function, Left/physiology , Blood Pressure , Coronary Vessels , Coronary Circulation/physiologySubject(s)
Lung Transplantation , Rodentia , Animals , Lung Transplantation/adverse effects , Gene Transfer Techniques , Genetic Therapy , LungABSTRACT
Shortage of donor organs for heart transplantation is a worldwide problem. Donation after circulatory death (DCD) has been proposed to expand the donor pool. However, in contrast to the donation after brain death that undergoes immediate cold preservation, warm ischemia and subsequent reperfusion injury are inevitable in DCD. It has been reported that interleukin-11 (IL-11) mitigates ischemia-reperfusion injury in rodent models of myocardial infarction and donation after brain death heart transplantation. We hypothesized that IL-11 also offers benefit to warm ischemia in an experimental model of cardiac transplantation that resembles DCD. The hearts of naïve male Sprague Dawley rats (n = 15/group) were procured, subjected to 25-min warm ischemia, and reperfused for 60 min using Langendorff apparatus. IL-11 or saline was administered intravenously before the procurement, added to maintenance buffer, and infused via perfusion during reperfusion. IL-11 group exhibited significantly better cardiac function post-reperfusion. Severely damaged mitochondria was found in the electron microscopic analysis of control hearts whereas the mitochondrial structure was better preserved in the IL-11 treated hearts. Immunoblot analysis using neonatal rat cardiomyocytes revealed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation at Ser727 after IL-11 treatment, suggesting its role in mitochondrial protection. Consistent with expected activation of mitochondrial respiration by mitochondrial STAT3, immunohistochemical staining demonstrated a higher mitochondrial cytochrome c oxidase subunit 2 expression. In summary, IL-11 protects the heart from warm ischemia reperfusion injury by alleviating mitochondrial injury and could be a viable therapeutic option for DCD heart transplantation.
Subject(s)
Heart Transplantation , Reperfusion Injury , Rats , Male , Animals , Humans , Interleukin-11/pharmacology , Brain Death , Rats, Sprague-Dawley , Reperfusion Injury/drug therapy , Reperfusion Injury/prevention & control , Tissue DonorsABSTRACT
It remains uncertain if body temperature (BT) is a useful prognostic indicator in coronavirus disease 2019 (COVID-19). We investigated the relationship between BT and mortality in COVID-19 patients. We used a de-identified database that prospectively collected information from patients screened for COVID-19 at the Mount Sinai facilities from February 28, 2020 to July 28, 2021. All patients diagnosed with COVID-19 that had BT data were included. BT at initial presentation, maximum BT during hospitalization, comorbidity, and vaccination status data were extracted. Mortality rate was assessed as a primary outcome. Among 24,293 cases, patients with initial BT below 36 °C had higher mortality than those with BT of 36-37 °C (p < 0.001, odds ratio 2.82). Initial BT > 38 °C was associated with high mortality with an incremental trend at higher BT. In 10,503 in-patient cases, a positive association was observed between mortality and maximum BT except in patients with BT < 36 °C. Multiple logistic regression analyses including the comorbidities revealed that maximum BT was an independent predictor of mortality. While vaccination did not change the distribution of maximum BT, mortality was decreased in vaccinated patients. Our retrospective cohort study suggests that high maximum BT is an independent predictor of higher mortality in COVID-19 patients.
Subject(s)
COVID-19 , Body Temperature , COVID-19/mortality , Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and overABSTRACT
Mechanical LV unloading for acute myocardial infarction (MI) is a promising supportive therapy to reperfusion. However, no data is available on exit strategy. We evaluated hemodynamic and cellular effects of reloading after Impella-mediated LV unloading in Yorkshire pigs. First, we conducted an acute study in normal heart to observe effects of unloading and reloading independent of MI-induced ischemic effects. We then completed an MI study to investigate optimal exit strategy on one-week infarct size, no-reflow area, and LV function with different reloading speeds. Initial studies showed that acute reloading causes an immediate rise in end-diastolic wall stress followed by a significant increase in cardiomyocyte apoptosis. The MI study did not result in any statistically significant findings; however, numerically smaller average infarct size and no-reflow area in the gradual reloading group prompt further examination of reloading approach as an important clinically relevant consideration.
ABSTRACT
BACKGROUND: SGLT2i (sodium-glucose cotransporter-2 inhibitors) improve clinical outcomes in patients with heart failure, but the mechanisms of action are not completely understood. SGLT2i increases circulating levels of ketone bodies, which has been demonstrated to enhance myocardial energetics and induce reverse ventricular remodeling. However, the role of SGLT2i or ketone bodies on myocardial ischemia reperfusion injury remains in the dark. The objective of this study is to investigate the cardioprotective potential of empagliflozin and ketone bodies during acute myocardial infarction (MI). METHODS: We used a nondiabetic porcine model of ischemia reperfusion using a percutaneous occlusion of proximal left anterior descending artery for 45 minutes. Animals received 1-week pretreatment with either empagliflozin or placebo prior to MI induction. Additionally, a third group received intravenous infusion of the ketone body BOHB (beta-hydroxybutyrate) during the MI induction. Acute effects of the treatments were assessed 4-hour post-MI by cardiac magnetic resonance and histology (thioflavin for area at risk, triphenyltetrazolium chloride staining for MI size). All animals were euthanized immediately postcardiac magnetic resonance, and heart samples were collected. RESULTS: The area at risk was similar in all groups. Empagliflozin treatment increased BOHB levels. Empagliflozin-treated animals showed significantly higher myocardial salvage, smaller MI size (both by cardiac magnetic resonance and histology), less microvascular obstruction, and improved cardiac function (left ventricle ejection fraction and strain). Furthermore, empagliflozin-treated animals demonstrated reduced biomarkers of cardiomyocyte apoptosis and oxidative stress compared with placebo. The BOHB group showed similar results to the empagliflozin group. CONCLUSIONS: One-week pretreatment with empagliflozin ameliorates ischemia reperfusion injury, reduces MI size and microvascular obstruction, increases myocardial salvage, preserves left ventricle systolic function, and lowers apoptosis and oxidative stress. Periprocedural intravenous infusion of BOHB during myocardial ischemia also induces cardioprotection, suggesting a role for BOHB availability as an additional mechanism within the wide spectrum of actions of SGLT2i.
Subject(s)
Myocardial Infarction , Sodium-Glucose Transporter 2 Inhibitors , Animals , Ketone Bodies/therapeutic use , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/therapeutic use , SwineABSTRACT
Impaired calcium uptake resulting from reduced expression and activity of the cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure (HF). Recently, new mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have emerged. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. SERCA2a is acetylated, and that acetylation is more prominent in failing human hearts. In this study, we confirmed that p300 interacts with and acetylates SERCA2a in cardiac tissues. Several lysine residues in SERCA2a modulated by p300 were identified using in vitro acetylation assay. Analysis of in vitro acetylated SERCA2a revealed several lysine residues in SERCA2a susceptible to acetylation by p300. Among them, SERCA2a Lys514 (K514) was confirmed to be essential for SERCA2a activity and stability using an acetylated mimicking mutant. Finally, the reintroduction of an acetyl-mimicking mutant of SERCA2a (K514Q) into SERCA2 knockout cardiomyocytes resulted in deteriorated cardiomyocyte function. Taken together, our data demonstrated that p300-mediated acetylation of SERCA2a is a critical PTM that decreases the pump's function and contributes to cardiac impairment in HF. SERCA2a acetylation can be targeted for therapeutic aims for the treatment of HF.
Subject(s)
Heart Failure , Protein Processing, Post-Translational , Sarcoplasmic Reticulum Calcium-Transporting ATPases , p300-CBP Transcription Factors , Humans , Heart Failure/metabolism , Lysine/metabolism , Myocytes, Cardiac/metabolism , p300-CBP Transcription Factors/chemistry , p300-CBP Transcription Factors/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolismABSTRACT
Despite significant advances in novel treatments and approaches, cardiovascular disease remains the leading cause of death globally. Gene therapy is a promising option for many diseases, including cardiovascular diseases. In the last 30 years, gene therapy has slowly proceeded towards clinical translation and recently reached US Food and Drug Administration approval for several diseases such as Leber congenital amaurosis and spinal muscular atrophy, among others. Previous attempts at developing gene therapies for cardiovascular diseases have yielded promising results in preclinical studies and early-phase clinical trials. However, larger trials failed to demonstrate consistent benefits in patients with ischemic heart disease and heart failure. In this review, we summarize the history and current status of clinical cardiac gene therapy. Starting with angiogenic gene therapy, we also cover more recent gene therapy trials for heart failure and cardiomyopathies. New programs are actively vying to be the first to get Food and Drug Administration approval for a cardiac gene therapy product by taking advantage of novel techniques.
Subject(s)
Cardiomyopathies , Cardiovascular Diseases , Heart Failure , Humans , Heart Failure/therapy , Genetic Therapy/methods , Enzyme InhibitorsABSTRACT
Coronary reperfusion therapy has played a pivotal role for reducing mortality and heart failure after acute myocardial infarction. Although several adjunctive approaches have been studied for reducing infarct size further, both ischemia-reperfusion injury and microvascular obstruction are still major contributors to both early and late clinical events after acute myocardial infarction. The progress in the field of cardioprotection has found several promising proof-of-concept preclinical studies. However, translation from bench to bedside has not been very successful. This comprehensive review discusses the importance of infarct size as a driver of clinical outcomes post-acute myocardial infarction and summarizes recent novel device-based approaches for infarct size reduction. Device-based interventions including mechanical cardiac unloading, myocardial cooling, coronary sinus interventions, supersaturated oxygen therapy, and vagal stimulation are discussed. Many of these approaches can modify ischemic myocardial biology before reperfusion and offer unique opportunities to target ischemia-reperfusion injury.
Subject(s)
Myocardial Infarction , Reperfusion Injury , Humans , Proof of Concept Study , Myocardial Infarction/therapyABSTRACT
Coronary atherosclerosis results from the delicate interplay of genetic and exogenous risk factors, principally taking place in metabolic organs and the arterial wall. Here we show that 224 gene-regulatory coexpression networks (GRNs) identified by integrating genetic and clinical data from patients with (n = 600) and without (n = 250) coronary artery disease (CAD) with RNA-seq data from seven disease-relevant tissues in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study largely capture this delicate interplay, explaining >54% of CAD heritability. Within 89 cross-tissue GRNs associated with clinical severity of CAD, 374 endocrine factors facilitated inter-organ interactions, primarily along an axis from adipose tissue to the liver (n = 152). This axis was independently replicated in genetically diverse mouse strains and by injection of recombinant forms of adipose endocrine factors (EPDR1, FCN2, FSTL3 and LBP) that markedly altered blood lipid and glucose levels in mice. Altogether, the STARNET database and the associated GRN browser (http://starnet.mssm.edu) provide a multiorgan framework for exploration of the molecular interplay between cardiometabolic disorders and CAD.
ABSTRACT
Gene therapy has made a significant progress in clinical translation over the past few years with several gene therapy products currently approved or anticipating approval for clinical use. Cardiac gene therapy lags behind that of other areas of diseases, with no application of cardiac gene therapy yet approved for clinical use. However, several clinical trials for gene therapy targeting the heart are underway, and innovative research studies are being conducted to close the gap. The second edition of Cardiac Gene Therapy in Methods in Molecular Biology provides protocols for cutting-edge methodologies used in these studies. In this chapter, we discuss recent updates on cardiac gene therapy studies and provide an overview of the chapters in the book.
Subject(s)
Dependovirus , Genetic Vectors , Dependovirus/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/genetics , HeartABSTRACT
Percutaneous antegrade coronary injection is among the least invasive cardiac selective gene delivery methods. However, the transduction efficiency of a simple bolus antegrade injection is quite low. In order to improve transduction efficiency in antegrade intracoronary delivery, several additional approaches have been proposed.In this chapter, we will describe the important elements associated with intracoronary delivery methods and present protocols for three different catheter-based antegrade gene delivery techniques in a preclinical large animal model. This is the second edition of this chapter, and it includes modifications we have made over the past several years that further enhance transduction efficacy.
Subject(s)
Gene Transfer Techniques , Genetic Therapy , Animals , HeartABSTRACT
Pulmonary hypertension (PH) is a devastating disease with high morbidity and mortality. Despite significant progress in the pharmacotherapy, current treatments only ameliorate the symptoms and cannot heal PH. Gene therapy may target the roots of the disease and holds evident promise. The current bottleneck for lung gene therapy is the delivery method. The requirements for the delivery mode are efficiency, safety, and the ability to target the anatomical site of interest, while avoiding off-target effects. Aerosolized gene delivery has been used in several studies and proven to be an efficient mode of administration for lung gene therapy. In this chapter, we describe a protocol of endobronchial aerosolization for PH gene therapy in a large animal model. Testing of a gene therapy in large animals is essential before clinical testing, since the lung anatomy and (patho)physiology differ immensely between humans and rodents, where most of the proof-of-concept studies are tested. The gene delivery vector is being aerosolized in the peripheral bronchi using a sprayer inserted through a flexible bronchoscope. This delivery mode results in efficient lung uptake and less off-target distribution relative to other airway delivery methods.
Subject(s)
Dependovirus , Hypertension, Pulmonary , Animals , Dependovirus/genetics , Disease Models, Animal , Genetic Therapy/methods , Genetic Vectors/genetics , Humans , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/therapyABSTRACT
The field of cardiac gene therapy has seen the rising use of adeno-associated viral (AAV) vectors as a promising therapeutic option for cardiac diseases and heart failure. To achieve intended results of AAV delivery, a majority of clinical studies screen patients for existing neutralizing antibodies that could inhibit the effects of the administered AAV and confound treatment efficacy. The cell-based neutralizing antibody assay offers a method of quantifying and identifying a patient's existing neutralizing antibodies against specific serotypes. Combined with the luciferase assay, the neutralizing antibody assay tests the ability of patient antibodies in the blood to prevent gene transduction of AAV-encoded luciferase gene at ranging serial dilutions. This chapter provides a protocol and experimental techniques to determine the presence of neutralizing antibodies against AAV in the blood.
Subject(s)
Antibodies, Neutralizing , Dependovirus , Antibodies, Viral , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Humans , SerogroupABSTRACT
Left ventricular (LV) catheterization with either a pressure catheter or pressure-volume catheter provides a means to measure cardiac function, an important endpoint in many studies including cardiac gene therapy. While the catheter can be inserted directly into the heart using a surgical approach, utilizing the carotid artery for access has the advantage of being a closed-chest procedure. This negates the need for intubation, prevents myocardial trauma, and preserves normal intrathoracic pressure, providing more accurate assessments of cardiac physiology parameters. We describe a protocol for obtaining carotid artery access and insertion of a pressure-volume catheter into the LV of rodents.