Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.

Sclerostin Downregulation Globally by Naturally Occurring Genetic Variants, or Locally in Atherosclerotic Plaques, Does Not Associate With Cardiovascular Events in Humans.

Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.

The Effects of Repeat-Dose Doxorubicin on Cardiovascular Functional Endpoints and Biomarkers in the Telemetry-Equipped Cynomolgus Monkey.

Doxorubicin-related heart failure has been recognized as a serious complication of cancer chemotherapy. This paper describes a cardiovascular safety pharmacology study with chronic dosing of doxorubicin in a non-human primate model designed to characterize the onset and magnitude of left ventricular dysfunction (LVD) using invasive and non-invasive methods. Cynomolgus monkeys ( = 12) were given repeated intravenous injections of doxorubicin over 135 days (19 weeks) with dosing holidays when there was evidence of significantly decreased hematopoiesis; a separate group ( = 12) received vehicle.

Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists.

Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability.

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response.

A bilayer small diameter vascular model for evaluation of drug induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury (DIVI) is defined as an adverse response to a drug characterized by degenerative and hyperplastic changes of endothelial cells and vascular smooth muscle cells. Inflammation may also be seen, along with extravasation of red blood cells into the smooth muscle layer (i.e.

High sensitivity flow cytometry of membrane vesicles.

Extracellular vesicles (EVs) are attracting attention as vehicles for inter-cellular signaling that may have value as diagnostic or therapeutic targets. EVs are released by many cell types and by different mechanisms, resulting in phenotypic heterogeneity that makes them a challenge to study. Flow cytometry is a popular tool for characterizing heterogeneous mixtures of particles such as cell types within blood, but the small size of EVs makes them difficult to measure using conventional flow cytometry.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

Objectives: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions.

Approach And Results: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins.

A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

Background: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype.

Methods: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues.

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl.

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level.

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month.

Integrated and translational nonclinical in vivo cardiovascular risk assessment: gaps and opportunities.

Cardiovascular (CV) safety concerns are a significant source of drug development attrition in the pharmaceutical industry today. Though current nonclinical testing paradigms have largely prevented catastrophic CV events in Phase I studies, many challenges relating to the inability of current nonclinical safety testing strategies to model patient outcomes persist. Contemporary approaches include a spectrum of evaluations of CV structure and function in a variety of laboratory animal species.

Current practices in preclinical drug development: gaps in hemostasis testing to assess risk of thromboembolic injury.

The Health and Environmental Sciences Institute Cardiac Biomarkers Working Group surveyed the pharmaceutical development community to investigate practices in assessing hemostasis, including detection of hypocoagulable and hypercoagulable states. Scientists involved in discovery, preclinical, and clinical research were queried on laboratory evaluation of endothelium, platelets, coagulation, and fibrinolysis during safety assessment studies. Results indicated that laboratory assessment of hemostasis is inconsistent among institutions and not harmonized between preclinical and clinical studies.

Exercise does not attenuate early CAD progression in a pig model.

Purpose: This study was designed to examine the effects of high-fat (HF) diet and subsequent exercise training (Ex) on coronary arteries of an animal model of early stage CAD. We hypothesized that HF diet would induce early stage disease and promote a proatherogenic coronary phenotype, whereas Ex would blunt disease progression and induce a healthier anti-inflammatory environment reflected by the increased expression of antioxidant capacity and the decreased expression of inflammatory markers in both the macrovasculature and the microvasculature of the coronary circulation.

Methods: Immunohistochemistry in left anterior descending and right coronary arteries and immunoblots in left anterior descending and left ventricular arterioles were used to characterize the effects of HF diet and Ex on the progression of coronary atherosclerosis.

A volumetric method for quantifying atherosclerosis in mice by using microCT: comparison to en face.

Precise quantification of atherosclerotic plaque in preclinical models of atherosclerosis requires the volumetric assessment of the lesion(s) while maintaining in situ architecture. Here we use micro-computed tomography (microCT) to detect ex vivo aortic plaque established in three dyslipidemic mouse models of atherosclerosis. All three models lack the low-density lipoprotein receptor (Ldlr(-/-)), each differing in plaque severity, allowing the evaluation of different plaque volumes using microCT technology.

Synthetic galectin-3 inhibitor increases metastatic cancer cell sensitivity to taxol-induced apoptosis in vitro and in vivo.

At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, beta-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway.

Comparison of distributions of survivin among tissues from urinary bladders of dogs with cystitis, transitional cell carcinoma, or histologically normal urinary bladders.

Objective: To compare distributions of survivin among tissues from urinary bladders of dogs with cystitis, transitional cell carcinoma (TCC), or histologically normal urinary bladders.

Sample Population: 24 archived and 7 fresh-frozen specimens of urinary bladders from dogs with cystitis.

Procedures: Immunohistochemical analysis of archived tissue specimens was performed to identify survivin protein in the nucleus and cytoplasm of cells by use of polyclonal rabbit anti-survivin antibody.

Exercise training alters effect of high-fat feeding on the ACTH stress response in pigs.

Eating and physical activity behaviors influence neuroendocrine output. The purpose of this study was to test, in an animal model of diet-induced cardiovascular disease, the effects of high-fat feeding and exercise training on hypothalamo-pituitary-adrenocortical (HPA) axis activity. We hypothesized that a high-fat diet would increase circulating free fatty acids (FFAs) and decrease the adrenocorticotropic hormone (ACTH) and cortisol response to an acute stressor.

PDGF/VEGF system activation and angiogenesis following initial post ovariectomy meningeal microvessel loss.

Recently we demonstrated that cessation of ovarian hormone production causes dramatic vascular remodeling in meningeal microvascular networks characterized by a significant microvessel loss. Further, even two months post ovariectomy (OVX), dura mater microvessels remain destabilized due to a decline in estrogen-mediated angiopoietin-1 (Ang-1) expression and Ang-1/Tie-2 signaling. Such destabilized microvessels could be susceptible to either further regression or angiogenesis.

Low-fat diet and exercise preserve eNOS regulation and endothelial function in the penis of early atherosclerotic pigs: a molecular analysis.

Introduction: Diet and exercise affect endothelial function in the penis, but the molecular mechanisms underlying their effects are not understood.

Aims: We evaluated endothelial nitric oxide synthase (eNOS) interaction with its negative regulator caveolin-1 and eNOS uncoupling as molecular targets in the penis associated with the beneficial effects of low-fat diet and chronic exercise.

Methods: The penes were obtained from adult male Yucatan pigs fed a normal-fat or high-fat diet on exercised or sedentary regimen for 24 weeks.