Vascular Responses and Biological Product Safety and Efficacy
Principal Investigator: Felice D'Agnillo, PhD
Office / Division / Lab: OBRR / DH / LBVB
The endothelial lining of the vascular system plays a key role in the regulation of hemostasis, inflammation, and vascular tone. Vascular endothelium, by virtue of its direct contact with the bloodstream, is a first line of defense against the potential adverse effects of circulating biological products or pathogens. The main focus of this research program is to study the mechanisms by which biological products or pathogens alter the functioning or integrity of the vascular system in both vascular endothelial cell culture systems and animal models. The overall goal of these studies is to improve the science-based regulation of current and anticipated products and to promote the design of novel or improved products and diagnostics. Our current research activities focus on two main areas: (1) Endothelial Dysfunction Triggered by Anthrax Toxin and (2) Markers of Hemoglobin Redox Activity and Toxicity.
Anthrax is a major bioterrorism threat to public health and security. Major initiatives in counterterrorism research were launched by the U.S. federal government in the aftermath of the 2001 anthrax attacks. Anthrax toxin, the main virulence factor of Bacillus anthracis, causes the severe illness and death associated with systemic anthrax infection. Post-exposure vaccination and antibiotics are not effective in treating the toxemia that occurs as the disease progresses. New anthrax therapies are therefore being developed to target the toxemia of an established infection. However, these efforts have been complicated by the fact that toxin-mediated pathogenesis is poorly understood. The goal of our research is to better define toxin-mediated pathogenesis and establish relevant biomarkers that will contribute to the evaluation and development of safe and effective anthrax therapies. Our research focuses on understanding the mechanisms underlying the vascular pathogenesis of anthrax using cell cultures as well as animal models.
Hemoglobin-based oxygen carriers or therapeutics (HBOCs) are being developed as red blood cell substitutes. Potential applications include resuscitation from hypovolemic shock, preoperative hemodilution for elective surgery, treatment of ischemic disease, and others. Given their desirable storage and compatibility characteristics, HBOCs may be useful in military settings and in situations that impact the blood supply such as major terrorist attacks or natural disasters. HBOCs have generally demonstrated promising safety and efficacy in animals and, in many cases, favorable Phase I clinical safety. However, the poor adverse event profile of some HBOCs in late phase clinical trials has led to suggestions that preclinical models may not have been sufficiently predictive of safety in humans. Moreover, concerns have been raised over the lack of sensitivity and specificity of standard histopathology and clinical chemistry indices for assessing the toxicity potential of this unique class of products. Our research program has focused on understanding the underlying mechanisms of hemoglobin toxicity using cell culture systems and animal models with the overall goal of improving the preclinical safety evaluation of HBOCs.
Our studies have directly supported the idea that vascular endothelium may be a key target for LT during systemic anthrax infection. Our findings have provided further insight on the mechanistic basis of LT-mediated barrier dysfunction. We have also reported LT enhances vascular cell adhesion molecule-1 (VCAM-1) expression and monocyte adhesion on tumor necrosis factor (TNF) activated primary human endothelial cells, suggesting a possible link between LT and the vasculitis associated with anthrax. The enhanced expression of VCAM-1 was found to be transcriptionally driven by the cooperative activation of the VCAM1-regulating transcription factors, interferon regulatory factor-1 (IRF-1) and NF-kB. LT enhancement of NF-kB activity was linked to enhanced IKK activation and differential IkB kinetics. Analysis of a number of different genes and transcription factors has also supported the important concept that LT can up- or down-regulate gene transcription depending on the cumulative effect of LT on the given set of transcription factors that regulate a particular gene. Taken together, these findings have significantly enhanced the understanding of toxin-mediated endothelial dysfunction and conceptually support the idea that endothelium may be a key therapeutic target especially during the established stages of the disease. These studies should provide useful paradigms for designing anthrax therapeutics aimed at directly neutralizing toxin or enhancing vascular endothelial protection against toxin. Such therapies could delay the rapid progression of the disease and extend the therapeutic window for clearing the bacteremia and toxemia and may be especially attractive as adjunct therapy.
The poor safety profiles of some HBOCs in late phase clinical trials has led to suggestions that preclinical testing may not have been sufficiently predictive of adverse events in humans. In this regard, the selection of relevant biomarkers may be critical for improving the predictability of preclinical studies. Using a 50% exchange transfusion model with polymerized bovine hemoglobin in rodents, we analyzed 4-hydroxynonenal (4-HNE)-modified protein adducts, an index of lipid peroxidation, 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, and cleaved caspase 3, a marker of apoptosis thought to be more sensitive than traditional TUNEL detection. Immunohistochemical and western blot analyses revealed enhanced accumulation of 4-HNE modified protein adducts in rat and guinea pig kidneys. Western blot analyses identified similar 4-HNE-modified protein adducts in lungs and hearts of guinea pigs. Nuclear 8-OHdG immunoreactivity was increased in renal proximal tubules and glomeruli in guinea pigs and co-localized with regions expressing high 4-HNE immunoreactivity. Cleaved caspase 3 was detectable by western blot in guinea pig and rat kidneys but was not associated with significant increases in serum creatinine or other common histopathological indices. Cleaved caspase 3 levels were also elevated in guinea pig hearts and lungs. The present findings suggest that sensitive and specific markers of oxidative stress and apoptosis may be useful in preclinical studies designed to evaluate the safety of these products.
Toxins 2014 Mar 31;6(4):1244-59
Extracellular hb enhances cardiac toxicity in endotoxemic Guinea pigs: protective role of haptoglobin.
Baek JH, Zhang X, Williams MC, Schaer DJ, Buehler PW, D'Agnillo F
PLoS One 2013 Apr 23;8(4):e62576
Anthrax lethal toxin downregulates claudin-5 expression in human endothelial tight junctions.
D'Agnillo F, Williams MC, Moayeri M, Warfel JM
J Clin Invest 2012 Apr 2;122(4):1444-58
Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy.
Baek JH, D'Agnillo F, Vallelian F, Pereira CP, Williams MC, Jia Y, Schaer DJ, Buehler PW
Toxins 2011 Oct;3(10):1278-93
Anthrax Lethal Toxin-Mediated Disruption of Endothelial VE-Cadherin Is Attenuated by Inhibition of the Rho-Associated Kinase Pathway.
Warfel JM, D'Agnillo F
Biochem Biophys Res Commun 2011 Jun 10;409(3):412-7
Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin.
Buehler PW, Butt OI, D'Agnillo F
Am J Pathol 2011 Mar;178(3):1316-28
Blood-brain barrier disruption and oxidative stress in Guinea pig after systemic exposure to modified cell-free hemoglobin.
Butt OI, Buehler PW, D'Agnillo F
Mucosal Immunol 2010 Sep;3(5):496-505
Progesterone regulation of uterine dendritic cell function in rodents is dependent on the stage of estrous cycle.
Butts CL, Candando KM, Warfel J, Belyavskaya E, D'Agnillo F, Sternberg EM
Trends Mol Med 2010 Aug 12;16(10):447-57
Hemoglobin-based oxygen carriers: from mechanisms of toxicity and clearance to rational drug design.
Buehler PW, D'Agnillo F, Schaer DJ
Antioxid Redox Signal 2010 Feb;12(2):199-208
Differential induction of renal heme oxygenase and ferritin in ascorbate and non-ascorbate producing species transfused with modified cell-free hemoglobin.
Butt OI, Buehler PW, D'Agnillo F
Antioxid Redox Signal 2010 Feb;12(2):275-91
Toxicological Consequences of Extracellular Hemoglobin: Biochemical and Physiological Perspectives.
Buehler PW, D'Agnillo F
J Biol Chem 2009 Sep 18;284(38):25761-71
Anthrax lethal toxin enhances IkappaB kinase activation and differentially regulates pro-inflammatory genes in human endothelium.
Warfel JM, D'Agnillo F
J Clin Invest 2009 Aug;119(8):2271-80
Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs.
Boretti FS, Buehler PW, D'Agnillo F, Kluge K, Glaus T, Butt OI, Jia Y, Goede J, Pereira CP, Maggiorini M, Schoedon G, Alayash AI, Schaer DJ
Biochem J 2008 Sep 15;414(3):461-9
Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes in haemodiluted rats.
Manalo DJ, Buehler PW, Baek JH, Butt O, D'agnillo F, Alayash AI
J Immunol 2008 Jun 1;180(11):7516-24
Anthrax Lethal Toxin Enhances TNF-Induced Endothelial VCAM-1 Expression via an IFN Regulatory Factor-1-Dependent Mechanism.
Warfel JM, D'Agnillo F
J Pharmacol Exp Ther 2007 Oct;323(1):49-60
Effects of endogenous ascorbate on oxidation, oxygenation and toxicokinetics of cell-free modified hemoglobin after exchange transfusion in rat and guinea pig.
Buehler PW, D'Agnillo F, Hoffman V, Alayash AI
Infect Immun 2007 Aug;75(8):3935-40
The large clostridial toxins from C. sordellii and C. difficile repress glucocorticoid receptor activity.
Tait AS, Dalton M, Geny B, D'Agnillo F, Popoff MR, Sternberg EM
Expert Opin Biol Ther 2007 May;7(5):665-75
First-generation blood substitutes: what have we learned? Biochemical and physiological perspectives.
Alayash AI, D'Agnillo F, Buehler PW
Biochem Biophys Res Commun 2005 Oct 7;337(4):1249-1256
Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells.
Steele AD, Warfel JM, D'Agnillo F
Am J Pathol 2005 Jun;166(6):1871-81
Anthrax lethal toxin induces endothelial barrier dysfunction.
Warfel JM, Steele AD, D'Agnillo F
Am J Physiol Heart Circ Physiol 2004 Oct;287(4):H1875-82
Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells.