Shawn Wagner

Collateral Artery Growth (Arteriogenesis) After Experimental Arterial Occlusion Is Impaired in Mice Lacking CC-Chemokine Receptor-2

Abstract— Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration–dependent migration, whereas this migration was abolished in cells from CCR2−/− mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2−/− mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45±0.06 and in CCR2−/− animals only to 0.21±0.03 (P <0.01). In C57BL/6 mice, ratio increased to 0.96±0.09 and 0.85±0.08 (P <0.05), respectively. MRI at 3 weeks (0.76±0.06 versus 0.62±0.01; P <0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2−/− mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2−/− mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2−/− animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

Impact of Mouse Strain Differences in Innate Hindlimb Collateral Vasculature

Objective—To assess the importance of genetic background for collateral artery development. Methods and Results—C57BL/6, BALB/c and 129S2/Sv mice were studied after femoral artery ligation by laser Doppler imaging, visible light oximetry, time-of-flight–magnetic resonance imaging, and treadmill testing; C57BL/6 and BALB/c also underwent electron paramagnetic resonance (EPR) oximetry, x-ray angiography, and histology. C57BL/6 had the least initial distal ischemia and most complete recovery. BALB/c had the most severe initial ischemia and poorest recovery. BALB/c had some vasodilatory reserve in their ligated limbs not seen in the other strains at 3 weeks. By in vivo TOF-magnetic resonance angiography, C57BL/6 had larger preexistent and developed collaterals. By x-ray angiography, C57BL/6 had a higher collateral-dependent filling score and number of visible collaterals immediately after femoral ligation and a higher number of visible collaterals at 1 week but not at 4 weeks. EPR oximetry and histology revealed hypoxia and tissue damage in regions of collateral growth of BALB/c but not C57BL/6 mice. In C57BL/6 BrdUrd uptake in the thigh was limited to larger vessels and isolated perivascular cells. Proliferative activity in collateral arterioles was similar in both strains. Conclusions—Genetic differences in preexistent collateral vasculature can profoundly affect outcome and milieu for compensatory collateral artery growth after femoral artery occlusion.

Angiopoietin-1 Promotes Tumor Angiogenesis in a Rat Glioma Model

Angiopoietins have been implicated in playing an important role in blood vessel formation, remodeling, maturation, and maintenance. However, the role of angiopoietins in tumor angiogenesis remains uncertain. In this study, expression of human angiopoietin-1 (hAng-1) and angiopoietin (hAng-2) was amplified in the rat glioma cell line GS9L by stable transfection using an inducible tet-off system. Transfected cells were implanted intracerebrally into syngenic Fischer 344 rats. We demonstrated by means of magnetic resonance imaging that increased hAng-1 expression promoted a significant in vivo growth of intracerebral gliomas in rats. Overexpression of hAng-1 resulted in more numerous, more highly branched vessels, which were covered by pericytes. On the other hand, tumors derived from hAng-2-overexpressing cells were smaller than empty-plasmid control tumors. The tumor vasculature in these tumors was composed of aberrant small vascular cords, which were associated with few mural cells. Our results indicate that in the presence of hAng-1, tumors induce a more functional vascular network, which led to better tumor perfusion and growth. On the other hand, overexpression of hAng-2 led to less intact tumor vessels, inhibited capillary sprouting, and impaired tumor growth.

Participation of Bone Marrow-Derived Cells in Long-Term Repair Processes after Experimental Stroke

Bone marrow-derived cells participate in remodeling processes of many ischemia-associated diseases, which has raised hopes for the use of bone marrow as a source for cell-based therapeutic approaches. To study the participation of bone marrow-derived cells in a stroke model, bone marrow from C57BL/6-TgN(ACTbEGFP)1Osb mice that express green fluorescent protein (GFP) in all cells was transplanted into C57BL/6J mice. The recipient mice underwent permanent occlusion of the middle cerebral artery, and bone marrow-derived cells were tracked by fluorescence. The authors investigated the involvement of bone marrow-derived cells in repair processes 6 weeks and 6 months after infarction. Six weeks after occlusion of the artery, more than 90% of the GFP-positive cells in the infarct border zone were microglial cells. Very few GFP-positive cells expressed endothelial markers in the infarct/infarct border zone, and no bone marrow-derived cells transdifferentiated into astrocytes, neurons, or oligodendroglial cells at all time points investigated. The results indicate the need for additional experimental studies to determine whether therapeutic application of nonselected bone marrow will replenish brain cells beyond an increase in microglial engraftment.

Parahydrogen-induced polarization (PHIP) hyperpolarized MR receptor imaging in vivo: a pilot study of 13C imaging of atheroma in mice.

MR techniques using hyperpolarized 13C have successfully produced examples of angiography and intermediary metabolic imaging, but, to date, no receptor imaging has been attempted. The goal of this study was to synthesize and evaluate a novel hyperpolarizable molecule, 2,2,3,3‐tetrafluoropropyl 1‐13C‐propionate‐d2,3,3 (TFPP), for the detection of atheromatous plaques in vivo. TFPP binds to lipid bilayers and its use in hyperpolarized MR could prove to be a major step towards receptor imaging. The precursor, 2,2,3,3‐tetrafluoropropyl 1‐13C‐acrylate‐d2,3,3 (TFPA), binds to 1,2‐dimyristoylphosphatidylcholine lipid bilayers with a 1.6‐ppm chemical shift in the 19F MR spectrum. This molecule was designed to be hyperpolarized through the addition of parahydrogen to the 13C‐acrylate moiety by parahydrogen‐induced polarization. TFPA was hyperpolarized to TFPP to an extent similar to that of the hydroxyethylacrylate to hydroxyethylpropionate transition: 17 ± 4% for TFPP versus 20% for hydroxyethylpropionate; T1 relaxation times (45 ± 2 s versus 55 ± 2 s) were comparable and the hyperpolarized properties of TFPP were characterized. Hydroxyethylacrylate, like TFPA, has a chemical structure with an acrylate moiety, but does not contain the lipid‐binding tetrafluoropropyl functional group. Hyperpolarized TFPP binds to the lipid bilayer, appearing as a second, chemically shifted 13C hyperpolarized MR signal with a further reduction in the longitudinal relaxation time (T1 = 21 ± 1 s). In aortas harvested from low‐density lipoprotein receptor knock‐out mice fed with a high‐fat diet for 9 months, and in which atheroma is deposited in the aorta and heart, TFPP showed greater binding to lipid on the intimal surface than in control mice fed a normal diet. When TFPP was hyperpolarized and administered in vivo to atheromatous mice in a pilot study, increased binding was observed on the endocardial surface of the intact heart compared with normally fed controls. Hyperpolarized TFPP has bio‐sensing specificity for lipid, coupled with a 42 000‐fold sensitivity gain in the MR signal at 4.7 T. Binding of TFPP with lipids results in the formation of a characteristic second peak in MRS. TFPP therefore has the potential to act as an in vivo molecular probe for atheromatous plaque imaging and may serve as a model of receptor‐targeted bio‐imaging with enhanced MR sensitivity. Copyright

Hyperpolarized MR Imaging: Neurologic Applications of Hyperpolarized Metabolism

SUMMARY: Hyperpolarization is the general term for a method of enhancing the spin-polarization difference of populations of nuclei in a magnetic field. No less than 5 distinct techniques (dynamic nuclear polarization [DNP]; parahydrogen-induced polarization−parahydrogen and synthesis allow dramatically enhanced nuclear alignment [PHIP-PASADENA]; xenon/helium polarization transfer; Brute Force; 1H hyperpolarized water) are currently under exhaustive investigation as means of amplifying the intrinsically (a few parts per million) weak signal intensity used in conventional MR neuroimaging and spectroscopy. HD-MR imaging in vivo is a metabolic imaging tool causing much of the interest in HD-MR imaging. The most successful to date has been DNP, in which carbon-13 (13C) pyruvic acid has shown many. PHIP-PASADENA with 13C succinate has shown HD-MR metabolism in vivo in tumor-bearing mice of several types, entering the Krebs−tricarboxylic acid cycle for ultrafast detection with 13C MR imaging, MR spectroscopy, and chemical shift imaging. We will discuss 5 promising preclinical studies: 13C succinate PHIP in brain tumor; 13C ethylpyruvate DNP and 13C acetate; DNP in rodent brain; 13C succinate PHIP versus gadolinium imaging of stroke; and 1H hyperpolarized imaging. Recent developments in clinical 13C neurospectroscopy encourage us to overcome the remaining barriers to clinical HD-MR imaging.

A Nanoparticle Catalyst for Heterogeneous Phase Para-Hydrogen-Induced Polarization in Water†

Para-hydrogen-induced polarization (PHIP) is a technique capable of producing spin polarization at a magnitude far greater than state-of-the-art magnets. A significant application of PHIP is to generate contrast agents for biomedical imaging. Clinically viable and effective contrast agents not only require high levels of polarization but heterogeneous catalysts that can be used in water to eliminate the toxicity impact. Herein, we demonstrate the use of Pt nanoparticles capped with glutathione to induce heterogeneous PHIP in water. The ligand-inhibited surface diffusion on the nanoparticles resulted in a (1) H polarization of P=0.25% for hydroxyethyl propionate, a known contrast agent for magnetic resonance angiography. Transferring the (1) H polarization to a (13) C nucleus using a para-hydrogen polarizer yielded a polarization of 0.013%. The nuclear-spin polarizations achieved in these experiments are the first reported to date involving heterogeneous reactions in water.

Time‐of‐flight quantitative measurements of blood flow in mouse hindlimbs

To evaluate the feasibility of using time‐of‐flight (TOF) imaging to directly measure hindlimb blood flow in a mouse model of peripheral vascular disease.

Inhibition of Collateral Artery Growth by Mibefradil: Possible Role of Volume-Regulated Chloride Channels

Endothelial cell swelling is one of the earliest hallmarks of arteriogenesis, the growth and maturation of collaterals. Mibefradil was found to block endothelial Cl(-) channels that control the volume of endothelial cells. Thus the authors investigated whether the blockade of volume-controlling endothelial cell channels would translate into an inhibition of arteriogenesis. In BALB/c mice, the right femoral artery was ligated and the animals received either mibefradil or solvent (phosphate-buffered saline [PBS]) via osmotic minipumps. Laser Doppler perfusion ratio (R/L) of ligated versus nonligated distal hindlimb increased from 0.06 +/- 0.01 (immediately after ligation) to 0.25 +/- 0.02 (day 7) in the PBS group and only from 0.07 +/- 0.02 to 0.13 +/- 0.02 in the mibefradil group (p <.01). Collateral artery diameters were significantly smaller in the mibefradil group (61 +/- 4.7 microm) versus controls (77.3 +/- 0.9 microm) (p <.05). Relative hemoglobin oxygen saturation measurements confirmed these findings (p <.02). The inhibition of arteriogenesis in the mibefradil group suggests that endothelial Cl(-) channels are involved in the initiation of arteriogenesis.

Curcumin Targeted, Polymalic Acid-Based MRI Contrast Agent for the Detection of Aβ Plaques in Alzheimer's Disease

Currently, there is no gadolinium-based contrast agent available for conventional magnetic resonance imaging (MRI) detection of amyloidal beta (Aβ) plaques in Alzheimers disease (AD). Its timely finding would be vital for patient survival and quality of life. Curcumin (CUR), a common Indian spice effectively binds to Aβ plaques which is a hallmark of AD. To address this binding, we have designed a novel nanoimaging agent (NIA) based on nature-derived poly(β-l-malic acid) (PMLA) containing covalently attached gadolinium-DOTA(Gd-DOTA) and nature-derived CUR. The all-in-one agent recognizes and selectively binds to Aβ plaques and is detected by MRI. It efficiently detected Aβ plaques in human and mouse samples by an ex vivo staining. The method can be useful in clinic for safe and noninvasive diagnosis of AD.