Quantitative assessment of myocardial blood flow and extracellular volume fraction using 68Ga-DOTA-PET: A feasibility and validation study in large animals.

BACKGROUND: Here we evaluated the feasibility of PET with Gallium-68 (68Ga)-labeled DOTA for non-invasive assessment of myocardial blood flow (MBF) and extracellular volume fraction (ECV) in a pig model of myocardial infarction. We also aimed to validate MBF measurements using microspheres as a gold standard in healthy pigs. METHODS: 8 healthy pigs underwent three sequential 68Ga-DOTA-PET/CT scans at rest and during pharmacological stress with simultaneous injection of fluorescent microspheres to validate MBF measurements. Myocardial infarction was induced in 5 additional pigs, which underwent 68Ga-DOTA-PET/CT examinations 7-days after reperfusion. Dynamic PET images were reconstructed and fitted to obtain MBF and ECV parametric maps. RESULTS: MBF assessed with 68Ga-DOTA-PET showed good correlation (y = 0.96x + 0.11, r = 0.91) with that measured with microspheres. MBF values obtained with 68Ga-DOTA-PET in the infarcted area (LAD, left anterior descendant) were significantly reduced in comparison to remote ones LCX (left circumflex artery, P < 0.0001) and RCA (right coronary artery, P < 0.0001). ECV increased in the infarcted area (P < 0.0001). CONCLUSION: 68Ga-DOTA-PET allowed non-invasive assessment of MBF and ECV in pigs with myocardial infarction and under rest-stress conditions. This technique could provide wide access to quantitative measurement of both MBF and ECV with PET imaging.

Sequential Bone-Marrow Cell Delivery of VEGFA/S1P Improves Vascularization and Limits Adverse Cardiac Remodeling After Myocardial Infarction in Mice.

Microvascular dysfunction and resulting tissue hypoxia is a major contributor to the pathogenesis and evolution of cardiovascular diseases (CVD). Diverse gene and cell therapies have been proposed to preserve the microvasculature or boost angiogenesis in CVD, with moderate benefit. This study tested in vivo the impact of sequential delivery by bone-marrow (BM) cells of the pro-angiogenic factors vascular endothelial growth factor (VEGFA) and sphingosine-1-phosphate (S1P) in a myocardial infarction model. For that, mouse BM cells were transduced with lentiviral vectors coding for VEGFA or sphingosine kinase (SPHK1), which catalyzes S1P production, and injected them intravenously 4 and 7 days after cardiac ischemia-reperfusion in mice. Sequential delivery by transduced BM cells of VEGFA and S1P led to increased endothelial cell numbers and shorter extravascular distances in the infarct zone, which support better oxygen diffusion 28 days post myocardial infarction, as shown by automated 3D image analysis of the microvasculature. Milder effects were observed in the remote zone, together with increased proportion of capillaries. BM cells delivering VEGFA and S1P also decreased myofibroblast abundance and restricted adverse cardiac remodeling without major impact on cardiac contractility. The results indicate that BM cells engineered to deliver VEGFA/S1P angiogenic factors sequentially may constitute a promising strategy to improve micro-vascularization and oxygen diffusion, thus limiting the adverse consequences of cardiac ischemia.

MT4-MMP deficiency increases patrolling monocyte recruitment to early lesions and accelerates atherosclerosis.

Matrix metalloproteinases are involved in vascular remodeling. Little is known about their immune regulatory role in atherosclerosis. Here we show that mice deficient for MT4-MMP have increased adherence of macrophages to inflamed peritonea, and larger lipid deposits and macrophage burden in atherosclerotic plaques. We also demonstrate that MT4-MMP deficiency results in higher numbers of patrolling monocytes crawling and adhered to inflamed endothelia, and the accumulation of Mafb+ apoptosis inhibitor of macrophage (AIM)+ macrophages at incipient atherosclerotic lesions in mice. Functionally, MT4-MMP-null Mafb+AIM+ peritoneal macrophages express higher AIM and scavenger receptor CD36, are more resistant to apoptosis, and bind acLDL avidly, all of which contribute to atherosclerosis. CCR5 inhibition alleviates these effects by hindering the enhanced recruitment of MT4-MMP-null patrolling monocytes to early atherosclerotic lesions, thus blocking Mafb+AIM+ macrophage accumulation and atherosclerosis acceleration. Our results suggest that MT4-MMP targeting may constitute a novel strategy to boost patrolling monocyte activity in early inflammation.

Assessment of regional pulmonary blood flow using 68Ga-DOTA PET.

BACKGROUND: In vivo determination of regional pulmonary blood flow (PBF) is a valuable tool for the evaluation of many lung diseases. In this study, the use of 68Ga-DOTA PET for the in vivo quantitative determination of regional PBF is proposed. This methodology was implemented and tested in healthy pigs and validated using fluorescent microspheres. The study was performed on young large white pigs (n = 4). To assess the reproducibility and consistency of the method, three PET scans were obtained for each animal. Each radiotracer injection was performed simultaneously to the injection of fluorescent microspheres. PBF images were generated applying a two-compartment exchange model over the dynamic PET images. PET and microspheres values were compared by regression analysis and Bland-Altman plot. RESULTS: The capability of the proposed technique to produce 3D regional PBF images was demonstrated. The correlation evaluation between 68Ga-DOTA PET and microspheres showed a good and significant correlation (r = 0.74, P < 0.001). CONCLUSIONS: Assessment of PBF with the proposed technique allows combining the high quantitative accuracy of PET imaging with the use of 68Ga/68Ge generators. Thus, 68Ga-DOTA PET emerges as a potential inexpensive method for measuring PBF in clinical settings with an extended use.

Cardiovascular imaging: what have we learned from animal models?

Cardiovascular imaging has become an indispensable tool for patient diagnosis and follow up. Probably the wide clinical applications of imaging are due to the possibility of a detailed and high quality description and quantification of cardiovascular system structure and function. Also phenomena that involve complex physiological mechanisms and biochemical pathways, such as inflammation and ischemia, can be visualized in a non-destructive way. The widespread use and evolution of imaging would not have been possible without animal studies. Animal models have allowed for instance, (i) the technical development of different imaging tools, (ii) to test hypothesis generated from human studies and finally, (iii) to evaluate the translational relevance assessment of in vitro and ex-vivo results. In this review, we will critically describe the contribution of animal models to the use of biomedical imaging in cardiovascular medicine. We will discuss the characteristics of the most frequent models used in/for imaging studies. We will cover the major findings of animal studies focused in the cardiovascular use of the repeatedly used imaging techniques in clinical practice and experimental studies. We will also describe the physiological findings and/or learning processes for imaging applications coming from models of the most common cardiovascular diseases. In these diseases, imaging research using animals has allowed the study of aspects such as: ventricular size, shape, global function, and wall thickening, local myocardial function, myocardial perfusion, metabolism and energetic assessment, infarct quantification, vascular lesion characterization, myocardial fiber structure, and myocardial calcium uptake. Finally we will discuss the limitations and future of imaging research with animal models.

Inhibition of proteasome by bortezomib causes intracellular aggregation of hepatic serpins and increases the latent circulating form of antithrombin.

Conformational diseases include heterogeneous disorders sharing a similar pathological mechanism, leading to intracellular aggregation of proteins with toxic effects. Serpins are commonly involved in these diseases. These are structurally sensitive molecules that modify their folding under even minor genetic or environmental variations. Indeed, under normal conditions, the rate of misfolding of serpins is high and unfolded serpins must be degraded by the proteasome system. Our aim was to study the effects of bortezomib, a proteasome inhibitor, on conformationally sensitive serpins. The effects of bortezomib were analysed in patients with multiple myeloma, HepG2 cells, and Swiss mice, as well as in vitro. Levels, anti-FXa activity, heparin affinity, and conformational features of antithrombin, a relevant anticoagulant serpin, were analysed. Histological, ultrastructural features and immunohistological distribution of antithrombin and alpha1-antitrypsin (another hepatic serpin) were evaluated. We also studied the intracellular accumulation of conformationally sensitive (fibrinogen) or non-sensitive (prothrombin) hepatic proteins. The inhibition of the proteasome caused intracellular accumulation and aggregation of serpins within the endoplasmic reticulum that was associated with confronting cisternae and Mallory body formation. These effects were accompanied by a heat stress response. Bortezomib also increased the levels of intracellular fibrinogen, but has no significant effect on prothrombin. Finally, bortezomib had only minor effects on the mature circulating antithrombin, with increased amounts of latent antithrombin in plasma. These results suggest that the impairment of proteasomal activities leads to an intracellular accumulation of conformationally sensitive proteins and might facilitate the release of misfolded serpins into circulation where they adopt more stable conformations.

Poly(ADP-ribose) polymerase-1 inhibition increases expression of heat shock proteins and attenuates heat stroke-induced liver injury.

OBJECTIVE: Heat stroke is a life-threatening illness characterized by an increased core body temperature as a result of exposure to high ambient temperature. Despite advances in supportive care, heat stroke is often fatal, and no specific and effective therapies exist. The pathophysiological responses to heat stroke involve a systemic inflammatory response and a disseminated intravascular coagulation in the host, which lead to a multiorgan dysfunction syndrome. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether inhibition of PARP activity might affect the heat stroke-induced injury. DESIGN: Controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: PARP-1-deficient mice (Parp-1(-/-)) and wild-type mice (C57BL/6J). INTERVENTIONS: Wild-type mice untreated or treated with either PJ34 or 3-AB, two generic PARP inhibitors, and Parp-1(-/-) mice were subjected to heat exposure as a model to study heat stroke. MEASUREMENTS AND MAIN RESULTS: We measured rectal temperature, serum interleukin-1beta and interleukin-6, liver histology, and heat shock proteins expression. We found that the heat stroke-induced injury was attenuated in mice lacking PARP-1 and was markedly reduced in wild-type mice treated with PARP inhibitors. Interestingly, heat-induced expression of heat shock proteins 27 and 70 was boosted after PARP inhibition. Indeed, PARP inhibition increased expression of heat shock proteins 27 and 70 even in the absence of heat exposure. Accordingly, PARP inhibition increased thermal tolerance that may contribute to attenuate the clinical effects of heat stroke, resulting in increased survival. CONCLUSIONS: Our results find a new protective function of PARP inhibitors and support their potential therapeutic application in the treatment of heat stroke.

Inhibition of poly(ADP-ribose) polymerase attenuates the severity of acute pancreatitis and associated lung injury.

The severity of acute pancreatitis results from the transmigration and activation of leukocytes within the pancreas and the local synthesis and release of proinflammatory-soluble mediators that transform a local injury into a systemic inflammatory response. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether the genetic deletion of PARP-1 or PARP-2 (a new member of the PARP family) genes, or pharmacological inhibition of PARP activity might affect the development and severity of acute pancreatitis and pancreatitis-associated lung injury. Secretagogue-induced acute pancreatitis was achieved by 12 hourly intraperitoneal injections of cerulein in mice deficient in PARP-1 or PARP-2 genes, and wild-type (WT) littermate mice untreated or treated with PARP activity inhibitors. The severity of pancreatitis was assessed by measurements of serum amylase, lipase, interleukin-1beta and IL-6, pancreatic water content, histologic grading and pancreas myeloperoxidase (MPO) activity. Lung injury was evaluated by quantifying MPO activity and morphological changes. We found that the severity of acute pancreatitis and pancreatitis-associated lung injury was significantly attenuated in mice lacking PARP-1, but not PARP-2, compared with WT mice. Interestingly, administration of PARP inhibitors, 3-aminobenzamide or PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl), in WT mice markedly decreased acute pancreatitis severity and pulmonary-associated injury in a larger extension than genetic deletion of PARP-1. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.