In vivo fluorescence imaging: success in preclinical imaging paves the way for clinical applications.

Advances in diagnostic imaging have provided unprecedented opportunities to detect diseases at early stages and with high reliability. Diagnostic imaging is also crucial to monitoring the progress or remission of disease and thus is often the central basis of therapeutic decision-making. Currently, several diagnostic imaging modalities (computed tomography, magnetic resonance imaging, and positron emission tomography, among others) are routinely used in clinics and present their own advantages and limitations. In vivo near-infrared (NIR) fluorescence imaging has recently emerged as an attractive imaging modality combining low cost, high sensitivity, and relative safety. As a preclinical tool, it can be used to investigate disease mechanisms and for testing novel diagnostics and therapeutics prior to their clinical use. However, the limited depth of tissue penetration is a major challenge to efficient clinical use. Therefore, the current clinical use of fluorescence imaging is limited to a few applications such as image-guided surgery on tumors and retinal angiography, using FDA-approved dyes. Progress in fluorophore development and NIR imaging technologies holds promise to extend their clinical application to oncology, cardiovascular diseases, plastic surgery, and brain imaging, among others. Nanotechnology is expected to revolutionize diagnostic in vivo fluorescence imaging through targeted delivery of NIR fluorescent probes using antibody conjugation. In this review, we discuss the latest advances in in vivo fluorescence imaging technologies, NIR fluorescent probes, and current and future clinical applications.

The pulmonary microvasculature entraps induced vascular progenitor cells (iVPCs) systemically delivered after cardiac ischemia-reperfusion injury: Indication for preservation of heart function via paracrine effects beyond engraftment.

OBJECTIVE: Stem cell-based regenerative therapies have been intensively studied with the aim to define an ideal cell type for the treatment of myocardial infarction. We tested systemically delivered, platelet-targeted induced vascular progenitor cells (iVPCs) to study their potential to salvage damaged myocardium after ischemia-reperfusion injury. METHODS: Using a mouse model of ischemia-reperfusion injury, we tested the potential of platelet-targeted iVPCs (1 × 106 targ-iVPCs) compared to non-targ-iVPCs and a saline control. Bioluminescence imaging, echocardiography, and histological analyses were performed. RESULTS: Four weeks after ischemia-reperfusion injury, systemic delivery of targ-iVPCs led to reduced fibrosis and infarct size (PBS: 25.7 ± 3.9 vs targ-iVPC: 18.4 ± 6.6 vs non-targ-iVPC: 25.1 ± 3.7%I/LV, P < 0.05), increased neovascularization, and restored cardiac function (PBS: 44.0 ± 4.2 vs targ-iVPC: 54.3 ± 4.5 vs non-targ-iVPC: 46.4 ± 3.8%EF, P < 0.01). Cell tracking experiments revealed entrapment of intravenously injected iVPCs in the pulmonary microvasculature in both cell-treated groups. CONCLUSIONS: Systemic delivery of iVPCs after cardiac ischemia-reperfusion injury is limited by pulmonary entrapment of the cells. Nevertheless, targ-iVPCs reduced infarct size, fibrosis, increased neovascularization, and most importantly retained cardiac function. These findings contribute to the mechanistic discussion of cell-based therapy and ultimately identify activated platelet-targeted iVPCs as candidates for cell therapy and also describe cell therapy benefits without the necessity of engrafting.

Absence of platelet endothelial cell adhesion molecule 1, PECAM-1/CD31, in vivo increases resistance to Salmonella enterica serovar Typhimurium in mice.

PECAM-1/CD31 is known to regulate inflammatory responses and exhibit pro- and anti-inflammatory functions. This study was designed to determine the functional role of PECAM-1 in susceptibility to murine primary in vivo infection with Salmonella enterica serovar Typhimurium and in in vitro inflammatory responses of peritoneal macrophages. Lectin profiling showed that cellular PECAM-1 and recombinant human PECAM-1-Ig chimera contain high levels of mannose sugars and N-acetylglucosamine. Consistent with this carbohydrate pattern, both recombinant human and murine PECAM-1-Ig chimeras were shown to bind S. Typhimurium in a dose-dependent manner in vitro. Using oral and fecal-oral transmission models of S. Typhimurium SL1344 infection, PECAM-1(-/-) mice were found to be more resistant to S. Typhimurium infection than wild-type (WT) C57BL/6 mice. While fecal shedding of S. Typhimurium was comparable in wild-type and PECAM-1(-/-) mice, the PECAM-1-deficient mice had lower bacterial loads in systemic organs such as liver, spleen, and mesenteric lymph nodes than WT mice, suggesting that extraintestinal dissemination was reduced in the absence of PECAM-1. This reduced bacterial load correlated with reduced tumor necrosis factor (TNF), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP) levels in sera of PECAM-1(-/-) mice. Following in vitro stimulation of macrophages with either whole S. Typhimurium, lipopolysaccharide (LPS) (Toll-like receptor 4 [TLR4] ligand), or poly(I·C) (TLR3 ligand), production of TNF and IL-6 by PECAM-1(-/-) macrophages was reduced. Together, these results suggest that PECAM-1 may have multiple functions in resistance to infection with S. Typhimurium, including binding to host cells, extraintestinal spread to deeper tissues, and regulation of inflammatory cytokine production by infected macrophages.

α-Tocopherol preserves cardiac function by reducing oxidative stress and inflammation in ischemia/reperfusion injury.

OBJECTIVE: Myocardial infarction (MI) is a leading cause of mortality and morbidity worldwide and new treatment strategies are highly sought-after. Paradoxically, reperfusion of the ischemic myocardium, as achieved with early percutaneous intervention, results in substantial damage to the heart (ischemia/reperfusion injury) caused by cell death due to aggravated inflammatory and oxidative stress responses. Chronic therapy with vitamin E is not effective in reducing the cardiovascular event rate, presumably through failing to reduce atherosclerotic plaque instability. Notably, acute treatment with vitamin E in patients suffering a MI has not been systematically investigated. METHODS AND RESULTS: We applied alpha-tocopherol (α-TOH), the strongest anti-oxidant form of vitamin E, in murine cardiac ischemia/reperfusion injury induced by ligation of the left anterior descending coronary artery for 60 min. α-TOH significantly reduced infarct size, restored cardiac function as measured by ejection fraction, fractional shortening, cardiac output, and stroke volume, and prevented pathological changes as assessed by state-of-the-art strain and strain-rate analysis. Cardioprotective mechanisms identified, include a decreased infiltration of neutrophils into cardiac tissue and a systemic anti-inflammatory shift from Ly6Chigh to Ly6Clow monocytes. Furthermore, we found a reduction in myeloperoxidase expression and activity, as well as a decrease in reactive oxygen species and the lipid peroxidation markers phosphatidylcholine (PC) (16:0)-9-hydroxyoctadecadienoic acid (HODE) and PC(16:0)-13-HODE) within the infarcted tissue. CONCLUSION: Overall, α-TOH inhibits ischemia/reperfusion injury-induced oxidative and inflammatory responses, and ultimately preserves cardiac function. Therefore, our study provides a strong incentive to test vitamin E as an acute therapy in patients suffering a MI.

Activated platelets in the tumor microenvironment for targeting of antibody-drug conjugates to tumors and metastases.

Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.

Author Correction: Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Comparison of MOLLI, shMOLLLI, and SASHA in discrimination between health and disease and relationship with histologically derived collagen volume fraction.

Aims: To determine the bioequivalence of several T1 mapping sequences in myocardial characterization of diffuse myocardial fibrosis. Methods and results: We performed an intra-individual sequence comparison of three types of T1 mapping sequences [MOdified Look-Locker Inversion recovery (MOLLI), Shortened MOdified Look-Locker Inversion recovery ((sh)MOLLI), and SAturation recovery single-SHot Acquisition (SASHA)]. We employed two model diseases of diffuse interstitial fibrosis [patients with non-ischaemic dilated cardiomyopathy (NIDCM), n = 32] and aortic stenosis [(AS), n = 25)]. Twenty-six healthy individuals served as controls. Relationship with collagen volume fraction (CVF) was assessed using endomyocardial biopsies (EMB) intraoperatively in 12 AS patients. T2 mapping (GraSE) was also performed. Myocardial native T1 with MOLLI and shMOLLI showed, firstly, an excellent discriminatory accuracy between health and disease [area under the curves (P-value): 0.94 (0.88-0.99); 0.87 (0.79-0.94); 0.61 (0.49-0.72)], secondly, relationship between histological CVF [native T1 MOLLI vs. shMOLLI vs. SASHA: r = 0.582 (P = 0.027), r = 0.524 (P = 0.046), r = 0.443 (P = 0.150)], and thirdly, with native T2 [r = 0.628(P < 0.001), r = 0.459 (P = 0.003), r = 0.211 (P = 0.083)]. The respective relationships for extracellular volume fraction with CVF [r = 0.489 (P = 0.044), r = 0.417 (0.071), r = 0.353 (P = 0.287)] were significant for MOLLI, but not other sequences. In AS patients, native T2 was significantly higher compared to controls, and associated with levels of C-reactive protein and troponin. Conclusion: T1 mapping sequences differ in their bioequivalence for discrimination between health and disease as well as associations with diffuse myocardial fibrosis.

Therapeutic targeting in nanomedicine: the future lies in recombinant antibodies.

The unique chemical and functional properties of nanoparticles can be harnessed for the delivery of large quantities of various therapeutic biomolecules. Active targeting of nanoparticles by conjugating ligands that bind to target cells strongly facilitates accumulation, internalization into target cells and longer retention at the target site, with consequent enhanced therapeutic effects. Recombinant antibodies with high selectivity and availability for a vast range of targets will dominate the future. In this review, we systematically outline the tremendous progress in the conjugation of antibodies to nanoparticles and the clear advantages that recombinant antibodies offer in the therapeutic targeting of nanoparticles. The demonstrated flexibility of recombinant antibody coupling to nanoparticles highlights the bright future of this technology for modern therapeutic nanomedicine.

A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology.

Progress in pharmaceutical development is highly-dependent on preclinical in vivo animal studies. Small animal imaging is invaluable for the identification of new disease markers and the evaluation of drug efficacy. Here, we report for the first time the use of a three-dimensional fluorescence bioimager called FLuorescence Emission Computed Tomography (FLECT) for the detection of a novel recombinant fluoroprobe that is safe, easily prepared on a large scale and stably stored prior to scan. This novel fluoroprobe (Targ-Cy7) comprises a single-chain antibody-fragment (scFvTarg), which binds exclusively to activated-platelets, conjugated to a near-infrared (NIR) dye, Cy7, for detection. Upon mouse carotid artery injury, the injected fluoroprobe circulates and binds within the platelet-rich thrombus. This specific in vivo binding of the fluoroprobe to the thrombus, compared to its non-targeting control-fluoroprobe, is detected by the FLECT imager. The analyzed FLECT image quantifies the NIR signal and localizes it to the site of vascular injury. The detected fluorescence is further verified using a two-dimensional IVIS® Lumina scanner, where significant NIR fluorescence is detected in vivo at the thrombotic site, and ex vivo, at the injured carotid artery. Furthermore, fluorescence levels in various organs have also been quantified for biodistribution, with the highest fluoroprobe uptake shown to be in the injured artery. Subsequently, this live animal imaging technique is successfully employed to monitor the response of the induced thrombus to treatment over time. This demonstrates the potential of using longitudinal FLECT scanning to examine the efficacy of candidate drugs in preclinical settings. Besides intravascular thrombosis, we have shown that this non-invasive FLECT-imaging can also detect in vivo pulmonary embolism. Overall, this report describes a novel fluorescence-based preclinical imaging modality that uses an easy-to-prepare and non-radioactive recombinant fluoroprobe. This represents a unique tool to study mechanisms of thromboembolic diseases and it will strongly facilitate the in vivo testing of antithrombotic drugs. Furthermore, the non-radiation nature, low-cost, high sensitivity, and the rapid advancement of optical scanning technologies make this fluorescence imaging an attractive development for future clinical applications.

Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging.

Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer using three different imaging modalities. These findings warrant further development of this activated platelet specific scFvGPIIb/IIIa, potentially as a universal marker for cancer diagnosis and ultimately for drug delivery in an innovative theranostic approach.

Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with 99mTechnecium-labelled recombinant complement receptor 2 (99mTc-rCR2), which specifically detects C3d at the site of complement activation. Compared to control imaging with an inactive CR2 mutant (99mTc-K41E CR2) or an irrelevant protein (99mTc-PSMA) or using 99mTc-rCR2 in C3-deficient mice, the use of 99mTc-rCR2 in complement-intact mice gave specific uptake in the reperfused myocardium. The heart to skeletal muscle ratio of 99mTc-rCR2 was significantly higher than in the three control groups. Histological analysis confirmed specific uptake of 99mTc-rCR2. Following therapeutic inhibition of complement C3 activation, we found reduced myocardial uptake of 99mTc-rCR2. We conclude, therefore that 99mTc-rCR2 imaging can be used for non-invasive detection of activated complement and in future could be exploited to quantify the severity of myocardial damage due to complement activation.