Project 1 Abstract We aim to understand the toxicities associated with the a cellular hemoglobin (Hb) in the circulation by applying in vivo microvascular tools and methods. Our results will expand the knowledge and therapeutic alternatives to hemolytic pathologies, blood transfusions and Hb-based oxygen carriers (HBOCs). Toxicity will be analyzed in terms of microcirculatory phenomena measured by quantifying changes in functional capillary density (FCD) in a hamster window chamber model. FCD is a general functional biomarker that integrates systemic, cardiovascular, cellular, and biochemical changes resulting from Hb-based transfusions. We will use Hbs generated by recombinant technology and chemical modifications that emphasize each of the properties that leads to a specific toxicity, namely: (a) tetramer dissociation, (b) oxygen transport, (c) scavenging of nitric oxide (NO), (d) oxidative degradation, (e) loss of heme component, and (f) solution rheology. The concentrations to be tested are in ranges associated with physiological conditions, i.e., trace amounts, top loads leading to concentrations found in hemolytic anemias, and in the range resulting from exchange-transfusion of HBOCs. Animal models with acute and chronic inflammation will be used to determine how to reveal the conditions of the circulation and particularly the endothelium influence the level of a cellular Hb induced toxicities. Different Hbs will be studied in normal animals at the three concentrations and in scenarios where a cellular Hb is likely to be present, namely: a) local tourniquet ischemia reperfusion (acute biochemically and mechanically induced vascular dysfunction), and, b) hemorrhagic shock resuscitation. A model using a high cholesterol ( 4 - 6 weeks, atherogenic) diet in hamsters will be used to analyze a cellular Hb toxicity conditions of chronic endothelial dysfunction. We will investigate the effects of blood rheological properties on the erythrocyte cell-free layer and how this affects vessel wall shear stress and vascular NO bioavailability. Project 2 will develop molecules with different O2 affinities, NO scavenging capacities, and resistances to oxidative degradation and hemin loss that will be tested with the goal of identifying properties that reduce toxicity. NO bioavailability will be manipulated by approaches that enhance vessel wall shear stress with increased viscosity, perfusion and co-infusion with NO releasing nanoparticles. Conjugation of Hb with haptoglobin will be used to control oxidative stress related toxicity. The effectiveness of reducing additives such as ascorbate will be tested. Our overall goal is to understand the relationships between Hb biophysical properties, acute stress, and vascular health on the severity of a cellular Hb induced toxicity to develop strategies to prevent or mitigate a cellular Hb toxicity.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Albert Einstein College of Medicine
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Meng, Fantao; Tsai, Amy G; Intaglietta, Marcos et al. (2015) PEGylation of ??-Hb using succinimidyl propionic acid PEG 5K: Conjugation chemistry and PEG shell structure dictate respectively the oxygen affinity and resuscitation fluid like properties of PEG ??-Hbs. Artif Cells Nanomed Biotechnol 43:270-81
Yalcin, Ozlem; Ortiz, Daniel; Tsai, Amy G et al. (2014) Microhemodynamic aberrations created by transfusion of stored blood. Transfusion 54:1015-27
Alayash, Abdu I (2014) Blood substitutes: why haven't we been more successful? Trends Biotechnol 32:177-85
Belcher, John D; Chen, Chunsheng; Nguyen, Julia et al. (2014) Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123:377-90
Liong, Celine; Ortiz, Daniel; Ao-ieong, Eilleen et al. (2014) Localized increase of tissue oxygen tension by magnetic targeted drug delivery. Nanotechnology 25:265102
Chatpun, Surapong; Cabrales, Pedro (2014) Nitric oxide synthase inhibition attenuates cardiac response to hemodilution with viscogenic plasma expander. Korean Circ J 44:105-12
Ortiz, Daniel; BriceƱo, Juan Carlos; Cabrales, Pedro (2014) Microhemodynamic parameters quantification from intravital microscopy videos. Physiol Meas 35:351-67
Mollan, Todd L; Jia, Yiping; Banerjee, Sambuddha et al. (2014) Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin. Free Radic Biol Med 69:265-77
Ortiz, Daniel; Barros, Marcelo; Yan, Su et al. (2014) Resuscitation from hemorrhagic shock using polymerized hemoglobin compared to blood. Am J Emerg Med 32:248-55
Sriram, Krishna; Intaglietta, Marcos; Tartakovsky, Daniel M (2014) Non-Newtonian flow of blood in arterioles: consequences for wall shear stress measurements. Microcirculation 21:628-39

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