Molecular imaging of cell death in vivo by a novel small molecule probe

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an 18F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

Purpose: Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. Methods: Using both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis. Results: ApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining. Conclusion: ApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states

Single administration of p2TA (AB103), a CD28 antagonist peptide, prevents inflammatory and thrombotic reactions and protects against gastrointestinal injury in total-body irradiated mice.

The goal of this study was to elucidate the action of the CD28 mimetic peptide p2TA (AB103) that attenuates an excessive inflammatory response in mitigating radiation-induced inflammatory injuries. BALB/c and A/J mice were divided into four groups: Control (C), Peptide (P; 5 mg/kg of p2TA peptide), Radiation (R; total body irradiation with 8 Gy γ-rays), and Radiation + Peptide (RP; irradiation followed by p2TA peptide 24 h later). Gastrointestinal tissue damage was evaluated by analysis of jejunum histopathology and immunohistochemistry for cell proliferation (Cyclin D1) and inflammation (COX-2) markers, as well as the presence of macrophages (F4/80). Pro-inflammatory cytokines IL-6 and KC as well as fibrinogen were quantified in plasma samples obtained from the same mice. Our results demonstrated that administration of p2TA peptide significantly reduced the irradiation-induced increase of IL-6 and fibrinogen in plasma 7 days after exposure. Seven days after total body irradiation with 8 Gy of gamma rays numbers of intestinal crypt cells were reduced and villi were shorter in irradiated animals compared to the controls. The p2TA peptide delivery 24 h after irradiation led to improved morphology of villi and crypts, increased Cyclin D1 expression, decreased COX-2 staining and decreased numbers of macrophages in small intestine of irradiated mice. Our study suggests that attenuation of CD28 signaling is a promising therapeutic approach for mitigation of radiation-induced tissue injury

Immunohistochemistry of activated macrophage marker F4/80 in mouse jejunum.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were immunostained with F4/80 antibodies and number of F4/80 positive cells per mm² area of tissue was counted using HistoQuest image analysis software. Bars represent the means ± SEMs for tissue sections of mice in each group. *Statistically significant differences between R and RP groups (P<0.05).</p

The effect of p<i>2TA</i> peptide on Cyclin D1 expression in mouse intestine.

<p>Expression of Cyclin D1 in mouse intestine: a–h) overview, i–p) details. a–d, i–l) A/J mice; e–h, m–p) BALB/c mice; a,e,i,m) sham irradiated controls; b,f,j,n) mice treated with p<i>2TA</i> peptide six days before tissue harvest; c,g,k,o) mice exposed to 8 Gy gamma rays total body irradiation seven days before the sacrifice; d,h,l,p) mice exposed to 8 Gy gamma rays that received 5 mg/kg p<i>2TA</i> peptide 24 hours after radiation exposure. In irradiated mice (c,g,k,o) villus integrity is disrupted, their crypts show disorganization, and very little Cyclin D1 staining can be observed outside the crypts. Intestines of irradiated mice that were treated with p<i>2TA</i> peptide 24 h later show a more normal morphology with respect to villus height and crypt appearance; they also show more numerous CD1 positive cells inside villi.</p

The effect of p<i>2TA</i> peptide on COX-2 expression in jejunal crypts.

<p>Expression of COX-2 in mouse intestine: a–h) overview, i–p) details. a–d, i–l) A/J mice; e–h, m–p) BALB/c mice; a,e,i,m) sham irradiated controls; b,f,j,n) mice treated with p<i>2TA</i> peptide six days before tissue harvest; c,g,k,o) mice exposed to 8 Gy gamma rays total body irradiation seven days before the sacrifice; d,h,l,p) mice exposed to 8 Gy gamma rays that received p<i>2TA</i> peptide 24 hours after radiation exposure. In irradiated mice (c,g,k,o) villus integrity is disrupted, their crypts show disorganization, and COX-2 staining is observable in cells in the surface layer of villi. Intestines of irradiated mice that were also treated with 5 mg/kg p<i>2TA</i> peptide 24 hours after irradiation show a more normal morphology with respect to villus height and crypt appearance, and they also show fewer COX-2 positive cells in villi surface cell layer, while COX-2 staining pattern inside villi replicates the pattern seen in sham irradiated mice.</p

The effect of p<i>2TA</i> peptide on jejunal crypts.

<p>Jejunal tissues were collected 7 days after irradiation and embedded in paraffin. After fixation and processing, cross sections of jejunum were stained with hematoxylin and eosin and analyzed for the number of surviving crypts (panel <b>A</b>) and villus height (panel <b>B</b>). Bars represent the means ± SEM of 4 cross-sections for each mouse, and 4–5 mice in each group. *Statistically significant difference between R and RP groups (P<0.05).</p

The effect of p<i>2TA</i> peptide on systemic inflammatory mediators.

<p>Plasma levels of IL-6 (panel <b>A</b>), KC (panel <b>B</b>) or fibrinogen (panel <b>C</b>) were measured on day 7 after irradiation. C: sham-irradiated mice, P: sham-irradiated mice that received 5 mg/kg of the p<i>2TA</i> peptide, R: 8 Gy-irradiated mice, RP: 8 Gy-irradiated mice that received 5 mg/kg of the p<i>2TA</i> peptide 24 h after irradiation. Bars represent the means ± SEM of 4–5 mice. *Statistically significant difference between R and RP groups (P<0.05).</p