Electrocardiogram-less, free-breathing myocardial extracellular volume fraction mapping in small animals at high heart rates using motion-resolved cardiovascular magnetic reesonance multitasking: a feasibility study in a heart failure with preserved ejection fraction rat model.

BACKGROUND: Extracellular volume fraction (ECV) quantification with cardiovascular magnetic resonance (CMR) T1 mapping is a powerful tool for the characterization of focal or diffuse myocardial fibrosis. However, it is technically challenging to acquire high-quality T1 and ECV maps in small animals for preclinical research because of high heart rates and high respiration rates. In this work, we developed an electrocardiogram (ECG)-less, free-breathing ECV mapping method using motion-resolved CMR Multitasking on a 9.4 T small animal CMR system. The feasibility of characterizing diffuse myocardial fibrosis was tested in a rat heart failure model with preserved ejection fraction (HFpEF). METHODS: High-salt fed rats diagnosed with HFpEF (n = 9) and control rats (n = 9) were imaged with the proposed ECV Multitasking technique. A 25-min exam, including two 4-min T1 Multitasking scans before and after gadolinium injection, were performed on each rat. It allows a cardiac temporal resolution of 20 ms for a heart rate of ~ 300 bpm. Myocardial ECV was calculated from the hematocrit (HCT) and fitted T1 values of the myocardium and the blood pool. Masson's trichrome stain was used to measure the extent of fibrosis. Welch's t-test was performed between control and HFpEF groups. RESULTS: ECV was significantly higher in the HFpEF group (22.4% ± 2.5% vs. 18.0% ± 2.1%, P = 0.0010). A moderate correlation between the ECV and the extent of fibrosis was found (R = 0.59, P = 0.0098). CONCLUSIONS: Motion-resolved ECV Multitasking CMR can quantify ECV in the rat myocardium at high heart rates without ECG triggering or respiratory gating. Elevated ECV found in the HFpEF group is consistent with previous human studies and well correlated with histological data. This technique has the potential to be a viable imaging tool for myocardial tissue characterization in small animal models.

Hyperpolarization of Amino Acids in Water Utilizing Parahydrogen on a Rhodium Nanocatalyst.

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para-hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13 C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology.

More Than 12 % Polarization and 20†Minute Lifetime of 15 N in a Choline Derivative Utilizing Parahydrogen and a Rhodium Nanocatalyst in Water.

Hyperpolarization techniques are key to extending the capabilities of MRI for the investigation of structural, functional and metabolic processes in vivo. Recent heterogeneous catalyst development has produced high polarization in water using parahydrogen with biologically relevant contrast agents. A heterogeneous ligand-stabilized Rh catalyst is introduced that is capable of achieving 15 N polarization of 12.2±2.7 % by hydrogenation of neurine into a choline derivative. This is the highest 15 N polarization of any parahydrogen method in water to date. Notably, this was performed using a deuterated quaternary amine with an exceptionally long spin-lattice relaxation time (T1 ) of 21.0±0.4 min. These results open the door to the possibility of 15 N in vivo imaging using nontoxic similar model systems because of the biocompatibility of the production media and the stability of the heterogeneous catalyst using parahydrogen-induced polarization (PHIP) as the hyperpolarization method.

Parahydrogen-Based Hyperpolarization for Biomedicine.

Magnetic resonance (MR) is one of the most versatile and useful physical effects used for human imaging, chemical analysis, and the elucidation of molecular structures. However, its full potential is rarely used, because only a small fraction of the nuclear spin ensemble is polarized, that is, aligned with the applied static magnetic field. Hyperpolarization methods seek other means to increase the polarization and thus the MR signal. A unique source of pure spin order is the entangled singlet spin state of dihydrogen, parahydrogen (pH2 ), which is inherently stable and long-lived. When brought into contact with another molecule, this "spin order on demand" allows the MR signal to be enhanced by several orders of magnitude. Considerable progress has been made in the past decade in the area of pH2 -based hyperpolarization techniques for biomedical applications. It is the goal of this Review to provide a selective overview of these developments, covering the areas of spin physics, catalysis, instrumentation, preparation of the contrast agents, and applications.

Aqueous Ligand-Stabilized Palladium Nanoparticle Catalysts for Parahydrogen-Induced 13C Hyperpolarization.

Parahydrogen-induced polarization (PHIP) is a method for enhancing NMR sensitivity. The pairwise addition of parahydrogen in aqueous media by heterogeneous catalysts can lead to applications in chemical and biological systems. Polarization enhancement can be transferred from 1H to 13C for longer lifetimes by using zero field cycling. In this work, water-dispersible N-acetylcysteine- and l-cysteine-stabilized palladium nanoparticles are introduced, and carbon polarizations up to 2 orders of magnitude higher than in previous aqueous heterogeneous PHIP systems are presented. P13C values of 1.2 and 0.2% are achieved for the formation of hydroxyethyl propionate from hydroxyethyl acrylate and ethyl acetate from vinyl acetate, respectively. Both nanoparticle systems are easily synthesized in open air, and TEM indicates an average size of 2.4 ± 0.6 nm for NAC@Pd and 2.5 ± 0.8 nm for LCys@Pd nanoparticles with 40 and 25% ligand coverage determined by thermogravimetric analysis, respectively. As a step toward biological relevance, results are presented for the unprotected amino acid allylglycine upon aqueous hydrogenation of propargylglycine.

Role of Interleukin-1 Signaling in a Mouse Model of Kawasaki Disease-Associated Abdominal Aortic Aneurysm.

OBJECTIVE: Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model. METHODS AND RESULTS: We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1ß mAb blocked LCWE-induced AAA formation. CONCLUSIONS: Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1ß play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

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.

Conversion rate of para-hydrogen to ortho-hydrogen by oxygen: implications for PHIP gas storage and utilization.

OBJECT: To determine the storability of para-hydrogen before reestablishment of the room temperature thermal equilibrium mixture. MATERIALS AND METHODS: Para-hydrogen was produced at near 100% purity and mixed with different oxygen quantities to determine the rate of conversion to the thermal equilibrium mixture of 75: 25% (ortho: para) by detecting the ortho-hydrogen (1)H nuclear magnetic resonance using a 9.4 T imager. RESULTS: The para-hydrogen to ortho-hydrogen velocity constant, k, near room temperature (292 K) was determined to be 8.27 ± 1.30 L/mol · min(-1). This value was calculated utilizing four different oxygen fractions. CONCLUSIONS: Para-hydrogen conversion to ortho-hydrogen by oxygen can be minimized for long term storage with judicious removal of oxygen contamination. Prior calculated velocity rates were confirmed demonstrating a dependence on only the oxygen concentration.

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

MR techniques using hyperpolarized (13)C 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-(13)C-propionate-d(2,3,3) (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-(13)C-acrylate-d(2,3,3) (TFPA), binds to 1,2-dimyristoylphosphatidylcholine lipid bilayers with a 1.6-ppm chemical shift in the (19)F MR spectrum. This molecule was designed to be hyperpolarized through the addition of parahydrogen to the (13)C-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; T(1) 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 (13)C hyperpolarized MR signal with a further reduction in the longitudinal relaxation time (T(1) = 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.

Intraoperative assessment and postsurgical treatment of prostate cancer tumors using tumor-targeted nanoprobes.

Successful visualization of prostate cancer (PCa) tumor margins during surgery remains a major challenge. The visualization of these tumors during surgery via near infrared fluorescence (NIRF) imaging would greatly enhance surgical resection, minimizing tumor recurrence and improving outcome. Furthermore, chemotherapy is typically administered to patients after surgery to treat any missed tumor tissue around the surgical area, minimizing metastasis and increasing patient survival. For these reasons, a theranostics fluorescent nanoparticle could be developed to assist in the visualization of PCa tumor margins, while also delivering chemotherapeutic drug after surgery. Methods: Ferumoxytol (FMX) conjugated to the fluorescent dye and PCa targeting agent, heptamethine carbocyanine (HMC), yielded the HMC-FMX nanoprobe that was tested in vitro with various PCa cell lines and in vivo with both subcutaneous and orthotopic PCa mouse models. Visualization of these tumors via NIRF imaging after administration of HMC-FMX was performed. In addition, delivery of chemotherapeutic drug and their effect on tumor growth was also assessed. Results: HMC-FMX internalized into PCa cells, labeling these cells and PCa tumors in mice with near infrared fluorescence, facilitating tumor margin visualization. HMC-FMX was also able to deliver drugs to these tumors, reducing cell migration and slowing down tumor growth. Conclusion: HMC-FMX specifically targeted PCa tumors in mice allowing for the visualization of tumor margins by NIRF imaging. Furthermore, delivery of anticancer drugs by HMC-FMX effectively reduced prostate tumor growth and reduced cell migration in vitro. Thus, HMC-FMX can potentially translate into the clinic as a nanotheranostics agent for the intraoperative visualization of PCa tumor margins, and post-operative treatment of tumors with HMC-FMX loaded with anticancer drugs.

Single- and Multi-Arm Gadolinium MRI Contrast Agents for Targeted Imaging of Glioblastoma.

BACKGROUND: Position of gadolinium atom(s) plays a key role in contrast enhancement of gadolinium-based contrast agents. To gain a better understanding of effects of distance of gadolinium in relation to the nanoconjugate platform, we designed and synthesized single- and multi-arm ("star") gadolinium conjugates equipped with antibody and peptides for targeting. The contrast agents were studied for their tumor imaging performance in a glioma mouse model. MATERIALS AND METHODS: Antibody- and peptide-targeted nano contrast agents (NCAs) were synthesized using polymalic acid platforms of different sizes. Gadolinium-DOTA and intermediates were attached as amides and targeting agents such as antibodies and peptides as thioethers. For in vivo experiments, we used human U87MG xenografts as glioma models. Magnetic resonance imaging (MRI) was performed on a Bruker BioSpec 94/20USR 9.4 T small-animal scanner. Delivery of contrast agents across the blood-brain barrier was studied by fluorescent microscopy. RESULTS: All contrast agents accumulated into tumor and showed composition-dependent imaging performance. Peptide-targeted mini-NCAs had hydrodynamic diameters in the range 5.2-9.4 nm and antibody-targeted NCAs had diameters in the range 15.8-20.5 nm. Zeta potentials were in the range of -5.4--8.2 mV and -4.6--8.8 mV, respectively. NCAs showed superior relaxivities compared to MultiHance at 9.4 T. The signal enhancement indicated maximum accumulation in tumor 30-60 minutes after intravenous injection of the mouse tail vein. Only targeted NCAs were retained in tumor for up to 3 hours and displayed contrast enhancement. CONCLUSION: The novel targeted NCAs with star-PEG features displayed improved relaxivity and greater contrast compared with commercial MultiHance contrast agent. The enhancement by mini-NCAs showed clearance of tumor contrast after 3 hours providing a suitable time window for tumor diagnosis in clinics. The technology provides a great tool with the promise of differential MRI diagnosis of brain tumors.

Near Infrared Fluorescent Nanoplatform for Targeted Intraoperative Resection and Chemotherapeutic Treatment of Glioblastoma.

Despite significant efforts to improve glioblastoma multiforme (GBM) treatment, GBM remains one of the most lethal cancers. Effective GBM treatments require sensitive intraoperative tumor visualization and effective postoperative chemotherapeutic delivery. Unfortunately, the diffusive and infiltrating nature of GBM limits the detection of GBM tumors, and current intraoperative visualization methods limit complete tumor resection. In addition, although chemotherapy is often used to eliminate any cancerous tissue remaining after surgery, most chemotherapeutic drugs do not effectively cross the brain-blood barrier (BBB) or enter GBM tumors. As a result, GBM has limited treatment options with high recurrence rates, and methods that improve its complete visualization during surgery and treatment are needed. Herein, we report a fluorescent nanoparticle platform for the near-infrared fluorescence (NIRF)-based tumor boundary visualization and image-guided drug delivery into GBM tumors. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved magnetic resonance imaging-sensitive superparamagnetic iron oxide nanoparticle, which is conjugated with hepthamethine cyanine (HMC), a NIRF ligand that specifically targets the organic anion transporter polypeptides that are overexpressed in GBM. We have shown that HMC-FMX nanoparticles cross the BBB and selectively accumulate in the tumor using orthotopic GBM mouse models, enabling NIRF-based visualization of infiltrating tumor tissue. In addition, HMC-FMX can encapsulate chemotherapeutic drugs, such as paclitaxel or cisplatin, and deliver these agents into GBM tumors, reducing tumor size and increasing survival. Taken together, these observations indicate that HMC-FMX is a promising nanoprobe for GBM surgical visualization and drug delivery.

In Vivo Imaging of Exogenous Progenitor Cells in Tendon Regeneration via Superparamagnetic Iron Oxide Particles.

BACKGROUND: Although tendon injuries and repairs are common, treatment of these injuries has limitations. The application of mesenchymal progenitor cells (MPCs) is increasingly used to optimize the biological process of tendon repair healing. However, clinically relevant technologies that effectively assess the localization of exogenous MPCs in vivo are lacking. HYPOTHESIS: Exogenous MPCs labeled with superparamagnetic iron oxide (SPIO) particles would allow monitoring of the localization and retention of cells within the site of implantation via magnetic resonance imaging (MRI) without negatively affecting cell survival or differentiation. STUDY DESIGN: Descriptive laboratory study. METHODS: Genetically modified C3H10T1/2 MPCs engineered to express luciferase (Luc+) reporter gene were implanted into surgically created Achilles tendon defects of 10 athymic nude rats (Hsd:RH-Foxn1rnu). Of these animals, 5 animals received Luc+ C3H10T1/2 MPCs colabeled with SPIO nanoparticles (+SPIO). These 2 groups of animals then underwent optical imaging with quantification of bioluminescence and MRI at 7, 14, and 28 days after surgery. Statistical analysis was conducted by use of 2-way analysis of variance. At 28 days after surgery, animals were euthanized and the treated limbs underwent histologic analysis. RESULTS: Optical imaging demonstrated that the implanted cells not only survived but also proliferated in vivo, and these cells remained viable for at least 4 weeks after implantation. In addition, SPIO labeling did not appear to affect MPC survival or proliferation, as assessed by quantitative bioluminescence imaging (P > .05, n = 5). MRI demonstrated that SPIO labeling was an effective method to monitor cell localization, retention, and viability for at least 4 weeks after implantation. Histologic and immunofluorescence analyses of the repaired tendon defect sites demonstrated tenocyte-like labeled cells, suggesting that cell differentiation was not affected by labeling the cells with the SPIO nanoparticles. CONCLUSION: MRI of exogenous MPCs labeled with SPIO particles allows for effective in vivo assessments of cell localization and retention in the setting of tendon regeneration for at least 4 weeks after implantation. This SPIO labeling does not appear to impair cell survival, transgene expression, or differentiation. CLINICAL RELEVANCE: SPIO labeling of MPCs appears to be safe for in vivo assessments of MPCs in tendon regeneration therapies and may be used for future clinical investigations of musculoskeletal regenerative medicine.

Assessing Therapeutic Efficacy in Real-time by Hyperpolarized Magnetic Resonance Metabolic Imaging.

Precisely measuring tumor-associated alterations in metabolism clinically will enable the efficient assessment of therapeutic responses. Advances in imaging technologies can exploit the differences in cancer-associated cell metabolism as compared to normal tissue metabolism, linking changes in target metabolism to therapeutic efficacy. Metabolic imaging by Positron Emission Tomography (PET) employing 2-fluoro-deoxy-glucose ([18F]FDG) has been used as a routine diagnostic tool in the clinic. Recently developed hyperpolarized Magnetic Resonance (HP-MR), which radically increases the sensitivity of conventional MRI, has created a renewed interest in functional and metabolic imaging. The successful translation of this technique to the clinic was achieved recently with measurements of 13C-pyruvate metabolism. Here, we review the potential clinical roles for metabolic imaging with hyperpolarized MRI as applied in assessing therapeutic intervention in different cancer systems.

Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4ß1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and ß1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.

Endothelial nitric oxide synthase activity is essential for vasodilation during blood flow recovery but not for arteriogenesis.

OBJECTIVE: Arteriogenesis is the major mechanism of vascular growth, which is able to compensate for blood flow deficiency after arterial occlusion. Endothelial nitric oxide synthase (eNOS) activity is essential for neovascularization, however its specific role in arteriogenesis remains unclear. We studied the role of eNOS in arteriogenesis using 3 mouse strains with different eNOS expression. METHODS AND RESULTS: Distal femoral artery ligation was performed in eNOS-overexpressing mice (eNOStg), eNOS-deficient (eNOS-/-) mice, and wild type (WT) controls. Tissue perfusion and collateral-dependent blood flow were significantly increased in eNOStg mice compared with WT only immediately after ligation. In eNOS-/- mice, although tissue perfusion remained significantly decreased, collateral-dependent blood flow was only decreased until day 7, suggesting normal, perhaps delayed collateral growth. Histology confirmed no differences in collateral arteries of eNOStg, eNOS-/-, and WT mice at 1 and 3 weeks. Administration of an NO donor induced vasodilation in collateral arteries of eNOS-/- mice, but not in WT, identifying the inability to vasodilate collateral arteries as main cause of impaired blood flow recovery in eNOS-/- mice. CONCLUSIONS: This study demonstrates that eNOS activity is crucial for NO-mediated vasodilation of peripheral collateral vessels after arterial occlusion but not for collateral artery growth.

Stromal epigenetic alterations drive metabolic and neuroendocrine prostate cancer reprogramming.

Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer-associated fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.

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.

Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2.

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.