Adelina Curaj

Repair After Myocardial Infarction, Between Fantasy and Reality The Role of Chemokines

Despite considerable progress over the last decades, acute myocardial infarction continues to remain the major cause of morbidity and mortality worldwide. The present therapies include only cause-dependent interventions, which are not able to reduce myocardial necrosis and optimize cardiac repair following infarction. This review highlights the cellular and molecular processes after myocardial injury and focuses on chemokines, the main modulators of the inflammatory and reparatory events, as the most valuable drug targets.

A New Monocyte Chemotactic Protein-1/ Chemokine CC Motif Ligand-2 Competitor Limiting Neointima Formation and Myocardial Ischemia/Reperfusion Injury in Mice

OBJECTIVES A nonagonist monocyte chemotactic protein-1 (MCP-1/CCL2) mutant (PA508) with increased affinity for glycosaminoglycans and thus competing with CCL2 was evaluated as a candidate for preventing neointima formation or myocardial ischemia/reperfusion injury. BACKGROUND Myocardial infarction (MI) remains a major cause of death worldwide despite improved interventional and therapeutic options. Therefore, the discovery of drugs that limit restenosis after intervention and post-MI damage remains an important challenge. METHODS The function of PA508 was assessed in functional assays in vitro and in mouse models of wire-induced neointima formation and experimental MI. RESULTS PA508 was functionally inactive in CC chemokine receptor 2 (CCR2) binding and calcium influx but inhibited monocyte chemotaxis or transendothelial migration toward CCL2, suggesting that it interferes with CCL2 presentation. In wild-type but not CCR2-deficient mice, PA508 reduced inflammatory leukocyte recruitment without affecting differential leukocyte counts, CCL2 levels, organ function, or morphology, indicating that it specifically attenuates the CCL2-CCR2 axis. Compared with vehicle, daily intraperitoneal injection of PA508 significantly (p < 0.05, n = 5) reduced neointimal plaque area and mononuclear cell infiltration in carotid arteries of hyperlipidemic apolipoprotein E-deficient mice while increasing smooth muscle cell content. In C57Bl/6J mice that underwent myocardial ischemia/reperfusion, treatment with PA508 significantly reduced infarction size, monocyte infiltration, and collagen and myofibroblast content in the infarction area and preserved heart function compared with vehicle (p < 0.05, n = 4 to 8). CONCLUSIONS Here we demonstrate that administration of a rationally designed CCL2 competitor reduced inflammatory monocyte recruitment, limited neointimal hyperplasia, and attenuated myocardial ischemia/reperfusion injury in mice and could therefore be envisioned as a combined therapeutic approach for restenosis and MI.

Synthesis, characterization antibacterial and antiproliferative activity of novel Cu(II) and Pd(II) complexes with 2-hydroxy-8-R-tricyclo[7.3.1.0.(2,7)]tridecane-13-one thiosemicarbazone.

Synthesis and biological activity investigation of complex compounds of Cu(II) are challenging issues because of the metal is not a xenobiotic one and the activity of ligands could be modulated by complexation. Complex combinations of Cu(II) and Pd(II) with thiosemicarbazone derivatives of 2-hydroxy-8-R-tricyclo[7.3.1.0.(2,7)]tridecane-13-one (where R=C(3)H(7), C(4)H(3)O) were synthesized. The characterization of the ligands and the newly formed compounds was done by (1)H NMR, (13)C NMR, UV-vis, IR, ESR spectroscopy, elemental analysis, molar electric conductibility and thermal studies. Experiments performed to identify the structures proved that the ligands coordinate to metal ions in different ways - neutral bidentate or mononegative bidentate. Also, if copper(II) acetate, copper(II) nitrate, copper(II) chloride and copper(II) thiocyanate were used, the ligands coordinated in a mononegative bidentate fashion. If copper(II) sulfate was used, the ligands coordinated in a neutral bidentate fashion. The biological activity for the copper(II) synthesized compounds was assessed in terms of antibacterial or antiproliferative activity. The antibacterial activity of the complexes against Staphylococcus aureus var. Oxford 6538, Escherichia coli ATCC 10536, Klebsielle pneumoniae ATCC 100131 and Candida albicans ATCC 10231 strains was studied and compared with that of free ligands. The effect of complex compounds on the proliferation of HeLa cells was tested. For all tested complexes an antiproliferative activity was noted at concentrations higher than 1 microM, but lower than 10 microM. Therefore, complex compounds of copper(II) were synthesized, structurally characterized and tested for biological activity, proving both antibacterial and antiproliferative activity.

Ultrasound molecular imaging of E-selectin in tumor vessels using poly n-butyl cyanoacrylate microbubbles covalently coupled to a short targeting peptide.

ObjectivesThe purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin–specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR. Materials and MethodsThe E-selectin–specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate–stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-&agr;–stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression. ResultsSuccess of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin–specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05). ConclusionsClinically translatable microbubbles that were covalently coupled to the short E-selectin–specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.

Noninvasive Molecular Ultrasound Monitoring of Vessel Healing After Intravascular Surgical Procedures in a Preclinical Setup

Objective—Cardiovascular interventions induce damage to the vessel wall making antithrombotic therapy inevitable until complete endothelial recovery. Without a method to accurately determine the endothelial status, many patients undergo prolonged anticoagulation therapy, denying them any invasive medical procedures, such as surgical operations and dental interventions. Therefore, we aim to introduce molecular ultrasound imaging of the vascular cell adhesion molecule (VCAM)-1 using targeted poly-n-butylcyanoacrylate microbubbles (MBVCAM-1) as an easy accessible method to monitor accurately the reendothelialization of vessels. Approach and Results—ApoE−/− mice were fed with an atherogenic diet for 1 and 12 weeks and subsequently, endothelial denudation was performed in the carotid arteries using a guidewire. Molecular ultrasound imaging was performed at different time points after denudation (1, 3, 7, and 14 days). An increased MBVCAM-1 binding after 1 day, a peak after 3 days, and a decrease after 7 days was found. After 12 weeks of diet, MBVCAM-1 binding also peaked after 3 days but remained high until 7 days, indicating a delay in endothelial recovery. Two-photon laser scanning microscopy imaging of double fluorescence staining confirmed the exposure of VCAM-1 on the superficial layer after arterial injury only during the healing phase. After complete reendothelialization, VCAM-1 expression persisted in the subendothelial layer but was not reachable for the MBVCAM-1 anymore. Conclusion—Molecular ultrasound imaging with MBVCAM-1 is promising to assess vascular damage and to monitor endothelial recovery after arterial interventions. Thus, it may become an important diagnostic tool supporting the development of adequate therapeutic strategies to personalize anticoagulant and anti-inflammatory therapy after cardiovascular intervention.

Rhodamine-Loaded Intercellular Adhesion Molecule–1-targeted Microbubbles for Dual-Modality Imaging Under Controlled Shear Stresses

Background—The ability to image incipient atherosclerosis is based on the early events taking place at the endothelial level. We hypothesized that the expression of intercellular adhesion molecule-1 even in vessels with high flow rates can be imaged at the molecular level using 2 complementary imaging techniques: 2-photon laser scanning microscopy and contrast-enhanced ultrasound. Methods and Results—Using 2-photon laser scanning microscopy and contrast-enhanced ultrasound, intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles were shown to be specifically bound to tumor necrosis factor-&agr;–stimulated human umbilical vein endothelial cells and murine carotid arteries (44 wild-type mice) at shear stresses ranging from 1.25 to 120 dyn/cm2. Intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles bound 8× more efficient (P=0.016) to stimulated human umbilical vein endothelial cells than to unstimulated cells and 14× more than nontargeted microbubbles (P=0.016). In excised carotids, binding efficiency did not decrease significantly when increasing the flow rate from 0.25 to 0.6 mL/min. Higher flow rates (0.8 and 1 mL/min) showed significantly reduced microbubbles retention, by 38% (P=0.03) and 55% (P=0.03), respectively. Ex vivo results were translatable in vivo, confirming that intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles are able to bind specifically to the inflamed carotid artery endothelia under physiological flow conditions and to be noninvasively detected using contrast-enhanced ultrasound. Conclusions—Our data provide groundwork for the implementation of molecular ultrasound imaging in vessels with high shear stress and flow rates, as well as for the future development of image-guided therapeutic interventions, and multiphoton microscopy as the appropriate method of validation.

Fluorescently labeled microbubbles for facilitating translational molecular ultrasound studies

Microbubbles (MB) are routinely used as contrast agents for functional and molecular ultrasound (US) imaging. For molecular US imaging, MB are functionalized with antibodies or peptides, in order to visualize receptor expression by angiogenic or inflamed endothelium. In general, initial in vitro binding studies with targeted MB are performed using phase contrast microscopy. Difficulties in the identification of MB in standard phase contrast microscopy, however, generally result in high variability, high observer dependency, and low reproducibility. To overcome these shortcomings, we here describe a simple post-loading strategy for labeling polymer-based MB with fluorophores, and we show that the use of rhodamine-loaded MB in combination with fluorescence microscopy substantially reduces the variability and the observer dependency of in vitro binding studies. In addition, we demonstrate that rhodamine-loaded MB can also be used for in vivo and ex vivo experimental setups, e.g., for analyzing MB binding to inflamed carotids using two-photon laser scanning microscopy, and for validating the binding of VEGFR2-targeted MB to tumor endothelium. These findings demonstrate that fluorescently labeled MB substantially facilitate translational molecular US studies, and they suggest that a similar synthetic strategy can be exploited for preparing drug-loaded MB, to enable image-guided, targeted, and triggered drug delivery to tumors and to sites of inflammation.

Novel insights into the mechanism of cell-based therapy after chronic myocardial infarction

Cell transplantation therapy is considered a novel and promising strategy in regenerative medicine. Recent studies point out that paracrine effects and inflammation induced by transplanted cells are key factors for the improvement of myocardial function. The present study aims at differentiating paracrine effects from inflammatory reactions after cell transplantation. Therefore, in vitro induced apoptotic bodies were transplanted after myocardial infarction in a rat model. Eight weeks after transplantation, the functional results showed no improvement in left ventricular function. Histological analysis revealed no significant differences in the amount of infiltrated cells and collagen content did not differ among the four groups, which sustains the functional data. Surprisingly, angiogenesis increased in groups with apoptotic bodies derived from HUVEC and endothelial progenitor cells, but not from fibroblasts. A complex genetic analysis of apoptotic bodies indicated that miRNAs could be responsible for these changes. Our study demonstrates that inflammatory reaction is critical for scar remodelling and improvement of the heart function after late cell therapy, while neoangiogenesis alone is not sufficient to improve heart function.

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

Blocking CCL5-CXCL4 heteromerization preserves heart function after myocardial infarction by attenuating leukocyte recruitment and NETosis

Myocardial infarction (MI) is a major cause of death in Western countries and finding new strategies for its prevention and treatment is thus of high priority. In a previous study, we have demonstrated a pathophysiologic relevance for the heterophilic interaction of CCL5 and CXCL4 in the progression of atherosclerosis. A specifically designed compound (MKEY) to block this CCL5-CXCR4 interaction is investigated as a potential therapeutic in a model of myocardial ischemia/reperfusion (I/R) damage. 8 week-old male C57BL/6 mice were intravenously treated with MKEY or scrambled control (sMKEY) from 1 day before, until up to 7 days after I/R. By using echocardiography and intraventricular pressure measurements, MKEY treatment resulted in a significant decrease in infarction size and preserved heart function as compared to sMKEY-treated animals. Moreover, MKEY treatment significantly reduced the inflammatory reaction following I/R, as revealed by specific staining for neutrophils and monocyte/macrophages. Interestingly, MKEY treatment led to a significant reduction of citrullinated histone 3 in the infarcted tissue, showing that MKEY can prevent neutrophil extracellular trap formation in vivo. Disrupting chemokine heterodimers during myocardial I/R might have clinical benefits, preserving the therapeutic benefit of blocking specific chemokines, and in addition, reducing the inflammatory side effects maintaining normal immune defence.