Copper Deficiency Induced Emphysema Is Associated with Focal Adhesion Kinase Inactivation

Background: Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1a) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein crosslinking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK). Methodology: To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA). Principal Findings: Copper depletion caused emphysematous changes, decreased HIF-1a activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim. Conclusions: These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema

Cerebral microbleeds in a neonatal rat model.

BACKGROUND:In adult humans, cerebral microbleeds play important roles in neurodegenerative diseases but in neonates, the consequences of cerebral microbleeds are unknown. In rats, a single pro-angiogenic stimulus in utero predisposes to cerebral microbleeds after birth at term, a time when late oligodendrocyte progenitors (pre-oligodendrocytes) dominate in the rat brain. We hypothesized that two independent pro-angiogenic stimuli in utero would be associated with a high likelihood of perinatal microbleeds that would be severely damaging to white matter. METHODS:Pregnant Wistar rats were subjected to intrauterine ischemia (IUI) and low-dose maternal lipopolysaccharide (mLPS) at embryonic day (E) 19. Pups were born vaginally or abdominally at E21-22. Brains were evaluated for angiogenic markers, microhemorrhages, myelination and axonal development. Neurological function was assessed out to 6 weeks. RESULTS:mRNA (Vegf, Cd31, Mmp2, Mmp9, Timp1, Timp2) and protein (CD31, MMP2, MMP9) for angiogenic markers, in situ proteolytic activity, and collagen IV immunoreactivity were altered, consistent with an angiogenic response. Vaginally delivered pups exposed to prenatal IUI+mLPS had spontaneous cerebral microbleeds, abnormal neurological function, and dysmorphic, hypomyelinated white matter and axonopathy. Pups exposed to the same pro-angiogenic stimuli in utero but delivered abdominally had minimal cerebral microbleeds, preserved myelination and axonal development, and neurological function similar to naïve controls. CONCLUSIONS:In rats, pro-angiogenic stimuli in utero can predispose to vascular fragility and lead to cerebral microbleeds. The study of microbleeds in the neonatal rat brain at full gestation may give insights into the consequences of microbleeds in human preterm infants during critical periods of white matter development

Correction: Cerebral microbleeds in a neonatal rat model.

[This corrects the article DOI: 10.1371/journal.pone.0171163.]

The effect of cerebral microbleeds on myelination.

<p><b>A–D</b>: Representative images (A–C), with quantification (D), of LFB staining (A) and MBP immunolabeling (B,C) at P52 in naïve pups with vaginal delivery (CTR-VD), pups following prenatal pro-angiogenic stimuli with vaginal delivery (PS-VD), and in pups following prenatal pro-angiogenic stimuli with abdominal delivery (PS-AD); arrows in (A) point to clumped myelinated fibers; arrows in (C) point to poorly myelinated fibers above corpus callosum; rectangles show ROI’s that were quantified; 7 rats/group; **, <i>p</i><0.01 comparing CTR-VD and PS-VD; §§, <i>p</i><0.01 comparing PS-VD and PS-AD; bars, 1 mm (A), 500 μm (B), 250 μm (C). <b>E</b>: Immunoblot (<i>left</i>), with quantification (<i>right</i>), of all bands of MBP at P52 in CTR-VD, PS-VD, and PS-AD rats; HSC70 used as loading control; 3 rats per group.</p

The effect of cerebral microbleeds on neurological function.

<p><b>A</b>: Performance on the righting reflex and on the negative geotaxis test on P3–14 in naïve pups with vaginal delivery (CTR-VD), pups following prenatal pro-angiogenic stimuli with vaginal delivery (PS-VD), and pups following prenatal pro-angiogenic stimuli with abdominal delivery (PS-AD). <b>B</b>: Performance on the open field test at P24, the elevated plus maze at P31, and on thigmotaxis at P35, in CTR-VD pups, PS-VD pups, and PS-AD pups. <b>C</b>: Spontaneous rearing at P31, performance on the beam balance test at P31, and grip strength at P31 in CTR-VD pups, PS-VD pups, and PS-AD pups. <b>D</b>: Incremental spatial learning on P35–39, performance on the memory probe at P40, and on the rapid learning test at P42 in CTR-VD pups, PS-VD pups, and PS-AD pups. For all panels, 19–25 pups/group; * and **, <i>p</i><0.05 and 0.01, respectively, comparing CTR-VD and PS-VD; §§, <i>p</i>< 0.01comparing PS-VD and PS-AD.</p

IUI+mLPS increases proteolytic activity and decreases collagen IV immunoreactivity.

<p><b>A–C</b>: <i>In situ</i> zymography (A,B), with quantification (C), of coronal sections from naïve control (CTR) and 24 hours after dual prenatal pro-angiogenic stimuli (PS) of IUI+mLPS, shown at low (A) and at high (B) magnification; the subventricular zone is shown in (B); nuclei stained with DAPI (blue); scale bars, 1 mm (A), 25 μm (B); 3 pups per group; *, <i>p</i><0.05; **, <i>p</i><0.01. <b>D,E</b>: Images of vessels identified by immunolabeling for RECA (red), that show proteolytic activity on <i>in situ</i> zymography (green); merged images are shown on the right; nuclei stained with DAPI (blue); scale bars, 50 μm. <b>F</b>: Immunolabeling for collagen IV (<i>left</i>), with quantification (<i>right</i>), on P0 in naïve controls (CTR), after IUI alone, after mLPS alone, and after the dual pro-angiogenic stimuli of IUI+mLPS (PS), in all cases after vaginal delivery, in coronal brain sections; 5 pups per group; tissues from the IUI alone group were from a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171163#pone.0171163.ref020" target="_blank">20</a>].</p

The effect of cerebral microbleeds on axonal development.

<p><b>A,B</b>: Representative images (A), with quantification (B), of immunolabeling for SIM-312 at P52 in naïve pups with vaginal delivery (CTR-VD), pups following prenatal pro-angiogenic stimuli with vaginal delivery (PS-VD), and in pups following prenatal pro-angiogenic stimuli with abdominal delivery (PS-AD); rectangle and oval show regions of interest (ROI) that were quantified; 7 rats/group; **, <i>p</i><0.01 comparing CTR-VD and PS-VD; §§, <i>p</i><0.01 comparing PS-VD and PS-AD; bar, 500 μm (A). <b>C</b>: Immunoblot (<i>left</i>), with quantification (<i>right</i>), of SIM-312 at P52 in CTR-VD, PS-VD, and PS-AD rats; HSC70 used as loading control; 3 rats per group.</p

qPCR primers used in this study.

<p>qPCR primers used in this study.</p