Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

Combined thrombogenic effects of vessel injury, pregnancy and procoagulant immune globulin administration in mice.

BACKGROUND: Pregnant women are at increased risk of thrombotic adverse events. Plasma derived immune globulin (IG) products, which are used in pregnancy for various indications, may contain procoagulant impurity activated coagulation factor XI (FXIa). Procoagulant IG products have been associated with increased thrombogenicity but their effect in pregnancy is unknown. METHODS: Late pregnant (gestation days 17-20) or early lactation (days 1-3) and control female mice were treated with IGs supplemented with human FXIa then subjected to ferric chloride (FeCl3) vessel injury. Occlusion of blood vessel was assessed by recording blood velocity in the femoral vein for 20 min using doppler ultrasound laser imaging. FXIa dose was selected by the ability to increase thrombin generation in mouse plasma in vitro. RESULTS: FXIa produced robust thrombin generation in mouse plasma ex vivo. Following FeCl3 injury, pregnant and non-pregnant mice receiving IG + FXIa exhibited faster reduction of blood velocity in femoral vein compared to IG alone or untreated controls. In vitro, thrombin generation in plasma samples collected after thrombosis in FXIa-treated animals was elevated and could be reduced by anti-FXI antibody. CONCLUSIONS: Our results suggest that intravenously-administered FXIa may contribute to thrombosis at the site of vascular injury in both pregnant and non-pregnant animals.

The protein phosphatase 2A B56γ regulatory subunit is required for heart development.

BACKGROUND: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made. RESULTS: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function. CONCLUSIONS: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits.

Cardiac-specific NRAP overexpression causes right ventricular dysfunction in mice.

The muscle-specific protein NRAP is concentrated at cardiac intercalated disks, plays a role in myofibril assembly, and is upregulated early in mouse models of dilated cardiomyopathy. Using a tet-off system, we developed novel transgenic lines exhibiting cardiac-specific NRAP overexpression ~2.5 times greater than normal. At 40-50 weeks, NRAP overexpression resulted in dilation and decreased ejection fraction in the right ventricle, with little effect on the left ventricle. Expression of transcripts encoding brain natriuretic peptide and skeletal α-actin was increased by cardiac-specific NRAP overexpression, indicative of a cardiomyopathic response. NRAP overexpression did not alter the levels or organization of N-cadherin and connexin-43. The results show that chronic NRAP overexpression in the mouse leads to right ventricular cardiomyopathy by 10 months, but that the early NRAP upregulation previously observed in some mouse models of dilated cardiomyopathy is unlikely to account for the remodeling of intercalated disks and left ventricular dysfunction observed in those cases.

The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia.

BACKGROUND: Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age-associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir-15a/16-1 locus, resulting in decreased mature miR-15a/16. METHODS: Spleens of aging NZB were analyzed for the presence of B-1 cells via flow cytometry and for the presence of a side population (SP) via the ability of cells to exclude Hoechst 33342 dye. The SP was assayed for the presence of hyperdiploid B-1 clones and for the ability to differentiate into B-1 cells in vitro and transfer disease in vivo. In addition, enhanced apoptosis of chemoresistant NZB B-1 cells was examined by restoring miR-16 levels in nutlin-treated cells. RESULTS: Aging NZB mice develop a B-1 expansion and clonal development that evolves from MBL into CLL. An expansion in SP is also seen. Although the SP did contain increased cells with stem cell markers, they lacked malignant B-1 cells and did not transfer disease in vivo. Similar to B-1 cells, splenic NZB SP also has decreased miR-15a/16 when compared with C57Bl/6. Exogenous addition of miR-15a/16 to NZB B-1 cells resulted in increased sensitivity to nutlin. CONCLUSION: NZB serve as an excellent model for studying the development and progression of age-associated CLL. NZB SP cells do not seem to contain cancer stem cells, but rather the B-1 stem cell. NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression.

Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation.

BACKGROUND: Establishing a large rodent model of brain metastasis that can be monitored using clinically relevant magnetic resonance imaging (MRI) techniques is challenging. Non-invasive imaging of brain metastasis in mice usually requires high field strength MR units and long imaging acquisition times. Using the brain seeking MDA-MB-231BR transfected with luciferase gene, a metastatic breast cancer brain tumor model was investigated in the nude rat. Serial MRI and bioluminescence imaging (BLI) was performed and findings were correlated with histology. Results demonstrated the utility of multimodality imaging in identifying unexpected sights of metastasis and monitoring the progression of disease in the nude rat. METHODS: Brain seeking breast cancer cells MDA-MB-231BR transfected with firefly luciferase (231BRL) were labeled with ferumoxides-protamine sulfate (FEPro) and 1-3 x 106 cells were intracardiac (IC) injected. MRI and BLI were performed up to 4 weeks to monitor the early breast cancer cell infiltration into the brain and formation of metastases. Rats were euthanized at different time points and the imaging findings were correlated with histological analysis to validate the presence of metastases in tissues. RESULTS: Early metastasis of the FEPro labeled 231BRL were demonstrated on T2*-weighted MRI and BLI within 1 week post IC injection of cells. Micro-metastatic tumors were detected in the brain on T2-weighted MRI as early as 2 weeks post-injection in greater than 85% of rats. Unexpected skeletal metastases from the 231BRL cells were demonstrated and validated by multimodal imaging. Brain metastases were clearly visible on T2 weighted MRI by 3-4 weeks post infusion of 231BRL cells, however BLI did not demonstrate photon flux activity originating from the brain in all animals due to scattering of the photons from tumors. CONCLUSION: A model of metastatic breast cancer in the nude rat was successfully developed and evaluated using multimodal imaging including MRI and BLI providing the ability to study the temporal and spatial distribution of metastases in the brain and skeleton.

Bone marrow-derived cells do not repair endothelium in a mouse model of chronic endothelial cell dysfunction.

AIMS: Bone marrow (BM)-derived endothelial progenitor cells (EPCs) in the circulation replace damaged vascular endothelium. We assessed the hypothesis that a BM transplant from healthy animals would restore normal arterial endothelium and prevent hypertension in young endothelial nitric oxide synthase-deficient (eNOS(-/-)) mice. METHODS AND RESULTS: Radiation or busulfan-induced BM ablation in eNOS(-/-) mice on day 6, day 14, or day 28 was followed by a BM transplant consisting of enhanced green fluorescent protein positive (EGFP(+)) cells from C57BL/6J mice. Peripheral blood cell chimerism was always greater than 85% at 4 months after BM transplant. Molecular assays of heart, kidney, and liver revealed low-level chimerism in all treatment groups, consistent with residual circulating EGFP(+) blood cells. When aorta, coronary, renal, hepatic, and splenic arteries in BM-transplanted eNOS(-/-) mice were examined by confocal microscopy, there were no EGFP- or eNOS-positive endothelial cells detected in these vessels in any of the treatment groups. Likewise, telemetry did not detect any reduction in blood pressure. Thus, no differences were observed in our measurements using several different treatment protocols. CONCLUSION: We found no evidence for BM-derived EPC renewal of endothelium in this eNOS-deficient mouse model of a chronic vascular disease or in wild-type mice during postnatal growth. Hence, renewal of chronic dysfunctional endothelium and endothelial homeostasis may be dependent on resident vascular progenitor cells.

Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress.

Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.

Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells.

Mutations in Notch receptors and their ligands have been identified as the cause of human congenital heart diseases, indicating the importance of the Notch signaling pathway during heart development. In our study, we use Cre-Lox technology to inactivate Notch2 in several cardiac cell lineages to determine the functional requirements for Notch2 during mammalian heart development. Inactivation of Notch2 in cardiac neural crest cells resulted in abnormally narrow aortas and pulmonary arteries due to a decrease in smooth muscle tissue. The reduction in smooth muscle tissue was not due to cell migration defects but instead was found to be caused by less proliferation in smooth muscle cells during mid to late gestation. Our findings demonstrate that Notch2 is required cell autonomously for proper formation of the heart outflow tract and provides insights into the role of Notch2 in vascular smooth muscle development and the cardiovascular defects associated with Alagille syndrome.

Rgs5 targeting leads to chronic low blood pressure and a lean body habitus.

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Comparison of Fenestra VC Contrast-enhanced computed tomography imaging with gadopentetate dimeglumine and ferucarbotran magnetic resonance imaging for the in vivo evaluation of murine liver damage after ischemia and reperfusion.

OBJECTIVES: Comparison of intravenous Fenestra VC-enhanced computed tomography (CT) with gadopentetate dimeglumine and Ferucarbotran contrast-enhanced magnetic resonance imaging (MRI) for the in vivo imaging of hepatic ischemia/reperfusion injury (IRI) in a murine model. MATERIAL AND METHODS: After induction of hepatic IRI by left liver lobe (LLL) ischemia (30, 45, and 75 minutes) and reperfusion (4 hours and 24 hours), a total of 130 mice were imaged either by Fenestra VC-enhanced 3-D CT or by dynamic, T1-weighed gadopentetate dimeglumine or static, T2*-weighed Ferucarbotran 2-D MRI (4.7 T). RESULTS: Detection of liver tissue damage as a consequence of IRI was not possible by CT or MRI without the use of contrast media. (1) Mice subjected to liver IRI (45 minutes of ischemia) and injected with Fenestra VC showed a distinct liver enhancement of the viable liver tissue or a nonenhancement of the necrotic tissue. The Fenestra VC CT-unenhanced liver volume increased as a function of time of ischemia and reperfusion. The unenhanced liver volume also correlated positively with serum liver enzyme activities and damage scores from liver histology. (2) The signal intensities (SI) between normal liver tissue and livers subjected to 30 minutes of ischemia were not different on dynamic gadopentetate dimeglumine-enhanced magnetic resonance images. More severe IRI as induced by 45 or 75 minutes of ischemia was characterized by (a) early hyperenhancement of regions in the LLL with rapid increase of SI higher than that observed in the undamaged liver within the first few minutes and (b) delayed hyperenhancement in the later course after gadopentetate dimeglumine injection, respectively. (3) Ferucarbotran MRI detected signs of IRI after only 30 minutes of liver ischemia and hence detected IRI earlier than Fenestra VC or gadopentetate dimeglumine. With longer duration of ischemia, Ferucarbotran SI increased in the LLL, but viable and necrotic tissues were not clearly distinguishable. CONCLUSIONS: MicroCT with Fenestra VC enhancement and MRI using either gadopentetate dimeglumine or Ferucarbotran enhancement of the liver revealed that all techniques allow in vivo determination of hepatic IRI as a function of the duration of ischemia and reperfusion of the liver. However, Fenestra VC-enhanced CT of the murine liver is superior to gadopentetate dimeglumine and Ferucarbotran for localization, quantification, and differentiation of viable from metabolically inactive/damaged liver tissue after hepatic ischemia/reperfusion but Fenestra VC is less sensitive than Ferucarbotran to detect the early onset of subtle consequences of hepatic IRI.