Induction of Shock After Intravenous Injection of Adenovirus Vectors: A Critical Role for Platelet-activating Factor  Zhili Xu, Jeffrey S. Smith, Jie Tian, Andrew P. Byrnes  Molecular Therapy  Volume 18, Issue 3, Pages 609-616 (March 2010) DOI: 10.1038/mt.2009.279 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Intravenous administration of Ad causes rapid hemoconcentration in rats. Sprague–Dawley rats were injected via the tail vein with adenovirus (Ad) vector at a dose of 1.2 × 1012 vp/kg and were killed at various time points. Blood was collected from the heart and the hematocrit was measured. Three to six rats per group. Nonreplicating E1-deleted Ad5 vectors were used in all experiments in this article. *P value of ≤0.05 versus control rats (Holm–Sidak post hoc). Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Ad induces PAF generation and causes shock in rats at an IV dose of 1.2 × 1012 vp/kg. (a) Blood levels of platelet-activating factor (PAF) were significantly elevated 5 minutes after intravenous (IV) injection of adenovirus (Ad). Three rats per group. An independent experiment yielded similar results. *P value of ≤0.05 (t-test). (b) Pretreatment with a PAF receptor antagonist prevented Ad-induced hemoconcentration. Five minutes before receiving Ad, rats were injected IV with saline or 0.1 mg/kg of the PAF receptor antagonist ABT-491. Ad was injected and 30 minutes later blood was collected for measurement of the hematocrit. Five rats per group. An independent experiment yielded similar results. *P value of ≤0.05 versus all other groups (Holm–Sidak post hoc). (c) Ad causes a rapid drop in systolic pressure that can be blocked with a PAF receptor antagonist. Anesthetized rats were preinjected IV with saline or ABT-491, and the tail pressure was monitored after IV injection of Ad. Ad induced severe hypotension in control rats, but ABT-491-pretreated rats were protected from hypotension and reproducibly demonstrated a brief hypertensive response to Ad. Injecting ABT-491 in the absence of Ad did not cause any change in blood pressure (data not shown). Results shown are representative of at least three different rats per group. The minimum detectable pressure with our tail-cuff apparatus was 60 mm Hg (dotted line); values below this level were not measurable. Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Systemic injection of Ad causes platelet-activating factor-dependent pathology in the duodenum and pancreas. Rats were predosed 5 minutes in advance with saline or ABT-491, followed by adenovirus (Ad) intravenous (IV) at 1.2 × 1012 vp/kg. (a) Ad tripled the hemoglobin content of the duodenum, and this response was blocked by pretreatment with ABT-491. Five or six rats per group. An independent experiment yielded similar results. *P value of ≤0.05 versus all other groups (Holm–Sidak post hoc). (b) Quantitation of the pancreatic weight before and after drying demonstrated that Ad-dependent edema in the pancreas was completely prevented by pretreatment with ABT-491. *P value of ≤0.05 versus all other groups (Holm–Sidak post hoc). Three to six rats per group. Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The RES is required for Ad to induce hemoconcentration and PAF. To deplete the reticuloendothelial system (RES), rats were injected intravenous (IV) with clodronate liposomes 1 day in advance. Rats were then injected IV with 1.2 × 1012 vp/kg of adenovirus (Ad). Alternatively, splenectomy or sham surgery was performed 2 weeks in advance. (a) Either clodronate treatment or splenectomy prevented Ad-induced hemoconcentration at 30 minutes. Two to four rats per group. *P value of ≤0.05 versus all other groups (Holm–Sidak post hoc). n.s. not significant. (b) Ad was unable to induce significant levels of platelet-activating factor (PAF) in clodronate-treated or splenectomized rats. PAF levels in the blood were measured 5 minutes after Ad dosing. Three or four rats per group. *P value of ≤0.05 versus buffer-injected rats (Holm–Sidak post hoc). Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Pretreating rats with a glucocorticoid partially protects against Ad-induced shock. (a) Dexamethasone reduced but did not eliminate the hematocrit response to adenovirus (Ad). Rats were preinjected subcutaneously with saline or 2 mg/kg dexamethasone, 2 hours in advance of Ad injection. Buffer or 1.2 × 1012 vp/kg of Ad was administered IV and blood was collected for hematocrit analysis at 30 minutes. Three or four rats per group. *P value of ≤0.05 (Holm–Sidak post hoc). (b) Dexamethasone did not protect rats from Ad-induced rapid hypotension. The tail pressure response to 1.2 × 1012 vp/kg of Ad was measured in anesthetized rats as described before. Results are representative of three rats per group. Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 PAF influences toxicity and expression in mice. (a) An intravenous (IV) adenovirus (Ad) dose of 1.2 × 1012 vp/kg did not cause hemoconcentration in mice. However, an Ad dose of 1.0 × 1013 vp/kg caused an elevated hematocrit, and this response could be prevented if mice were preinjected 5 minutes in advance with ABT-491 (0.1 mg/kg IV). The hematocrit was measured 30 minutes after Ad dosing. Four C57BL/6 mice per group. *P value of ≤0.05 versus all other groups (Holm–Sidak post hoc). (b) ABT-491 had a beneficial effect on Ad-mediated luciferase expression in the liver, with a sixfold increase in expression at the low dose of Ad and a twofold increase at the high dose of Ad. Luciferase expression was measured at 48 hours (n = 5 mice per group). *P value of ≤0.05 (Holm–Sidak post hoc). (c) ABT-491 did not significantly affect the amount of Ad DNA in the liver at 48 hours, measured in the same liver samples as in b. (d) ABT-491 did not protect mice from Ad-induced liver damage. Blocking platelet-activating factor (PAF) receptors with ABT-491 actually resulted in a significant increase in plasma levels of the liver enzyme ALT, measured 24 hours after an Ad dose of 1.0 × 1013 vp/kg (n = 5 mice per group). Treating mice with ABT-491 by itself did not cause any change in baseline levels of ALT (data not shown). *P value of ≤0.05 (Holm–Sidak post hoc). Molecular Therapy 2010 18, 609-616DOI: (10.1038/mt.2009.279) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions