Background NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived ROS in the myocardium. We investigated the role of Nox4 and Nox4 associated signaling pathways in the development of cardiac remodeling. Methods and Results Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: 1) control mice (CTL): littermates that are negative for hNox4 transgene but Cre positive; 2) c-hNox4 Tg mice; 3) angiotensin II (AngII)-infused CTL mice and 4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited approximately 10-fold increase in Nox4 protein expression and 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII-infusion to CTL mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least partially by enhancing Nox4 expression. Moreover, hNox4 transgene and/or AngII-infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed Akt-mTOR and NFκB signaling pathway and attenuated cardiac remodeling. Conclusion Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling.

Abstract-Angiotensin II (Ang II) upregulates vascular endothelial growth factor (VEGF) and activates vascular inflammation. However, the decisive role of VEGF in Ang II-induced vascular inflammation and remodeling has not been addressed. Ang II infusion to wild-type mice increased local expression of VEGF and its receptors in cells of aortic wall and plasma VEGF, and caused aortic inflammation (monocyte infiltration) and remodeling (wall thickening and fibrosis).

Previous MRI studies confirmed abnormalities in the limbic-cortical-striatal-pallidal-thalamic (LCSPT) network or limbic-cortico-striatal-thalamic-cortical (LCSTC) circuits in patients with major depressive disorder (MDD), but few studies have investigated the subcortical structural abnormalities. Therefore, we sought to determine whether focal subcortical grey matter (GM) changes might be present in MDD at an early stage. We recruited 30 first episode, untreated patients with major depressive disorder (MDD) and 26 healthy control subjects. Voxel-based morphometry was used to evaluate cortical grey matter changes, and automated volumetric and shape analyses were used to assess volume and shape changes of the subcortical GM structures, respectively. In addition, probabilistic tractography methods were used to demonstrate the relationship between the subcortical and the cortical GM. Compared to healthy controls, MDD patients had significant volume reductions in the bilateral putamen and left thalamus (FWE-corrected, p < 0.05). Meanwhile, the vertex-based shape analysis showed regionally contracted areas on the dorsolateral and ventromedial aspects of the bilateral putamen, and on the dorsal and ventral aspects of left thalamus in MDD patients (FWE-corrected, p < 0.05). Additionally, a negative correlation was found between local atrophy in the dorsal aspects of the left thalamus and clinical variables representing severity. Furthermore, probabilistic tractography demonstrated that the area of shape deformation of the bilateral putamen and left thalamus have connections with the frontal and temporal lobes, which were found to be related to major depression. Our results suggested that structural abnormalities in the putamen and thalamus might be present in the early stages of MDD, which support the role of subcortical structure in the pathophysiology of MDD. Meanwhile, the present study showed that these subcortical structural abnormalities might be the potential trait markers of MDD.

It has been presupposed that the thermodynamic stability constant ( Ktherm) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low- Ktherm gadodiamide with high- Ktherm gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low- Ktherm chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.

Objective-Vascular endothelial growth factor (VEGF) is upregulated after arterial injury. Its role in the pathogenesis of neointimal formation after periadventitial injury, however, has not been addressed. Methods and Results-Expression of VEGF and its receptors but not that of placental growth factor markedly increased with the development of neointimal formation in hypercholesterolemic mice after cuff-induced periarterial injury. Transfection with the murine soluble Flt-1 (sFlt-1) gene to block VEGF in vivo in mice inhibited early inflammation and later neointimal formation. The sFlt-1 gene transfer did not affect plasma lipid levels but attenuated increased expression of VEGF, Flt-1, Flk-1, monocyte chemoattractant protein-1, and other inflammation-promoting factors. Mice with Flt-1 kinase deficiency also displayed reduced neointimal formation. Key Words: remodeling Ⅲ growth substances Ⅲ inflammation Ⅲ arteriosclerosis Ⅲ gene therapy N eointimal formation is a major cause of restenosis after coronary intervention. 1,2 Vascular endothelial growth factor (VEGF) and its receptors (VRGFR-1: Flt-1, VEGFR-2: Flk-1) are upregulated in vascular inflammatory and proliferative disorders such as atherosclerosis and restenosis. [3][4][5][6] VEGF is thought to protect the artery from such disorders by inducing endothelial regeneration and improving endothelial function. 7 VEGF gene transfer or administration of its protein induces endothelial regeneration and attenuates neointimal formation after endothelial injury. 7-9 VEGF is reported to inhibit leukocyte infiltration through hemeoxygenase-1. 10 There is still considerable debate, however, over the role of VEGF in the development of neointimal formation after injury. 11,12 Emerging evidence suggests that VEGF causes or promotes the development of atherosclerosis or neointimal formation after injury. VEGF induces migration and activation of monocytes, 13 adhesion molecules, 14 or monocyte chemoattractant protein-1 (MCP-1) 15 through its receptor Flt-1. Moreover, administration of VEGF protein to hypercholesterolemic animals enhances atherogenesis by inducing monocyte infiltration and activation. 16 In addition, VEGF might promote migration of vascular smooth muscle cells though 18 Angiogenesis inhibitors are shown to reduce intimal neovascularization and plaque growth in hyperlipidemic mice. 19 One major reason for the inconsistent reports regarding the role of VEGF might be because there are no selective VEGF inhibitors tested. The only known endogenous VEGF inhibitor is a soluble form of the VEGF receptor-1, Flt-1 (sFlt-1). 20 This isoform is mainly expressed by vascular endothelial cells and can inhibit VEGF activity by directly sequestering VEGF and by functioning as a dominant-negative inhibitor. 20 We and others previously demonstrated that intramuscular transfection of the sFlt-1 gene blocks VEGF signaling and thus quenches VEGF activity in vivo. 21,22 Therefore, sFlt-1 gene transfer can be used as an inhibitor against VEGF and its receptors (Flt-1, Flk-1). In add...

Evidence for gadolinium-based contrast agent-(GBCA-) induced disease continues to mount. Risk factors for gadolinium-induced systemic fibrosis are entirely unexplored. Obesity-related renal injury is characterized by activation of glomerular mesangial cells and podocyte damage with alteration of lipid metabolism/lipid accumulation in both cell types resulting in matrix accumulation and eventual progression to glomerulosclerosis. We examined the consequences of GBCA treatment in the kidneys from mice with normal kidney function and the potential interplay between obesity and gadolinium exposure. We found that administration of GBCA (4 weeks) causes significant renal fibrosis and podocyte injury that are associated with metabolic disorders as evidenced by dyslipidemia. Metabolomic analysis demonstrated that renal lipid metabolism and metabolic markers of collagen turnover are significantly altered by gadolinium. GBCA stimulates myeloid-derived fibrocytes to the kidney. Obesity was induced by feeding a group of mice a high fat diet (HFD) for 22 weeks. Groups were sub-randomized to GBCA treatment versus none for 4 weeks before sacrifice. HFD-induced fibrosis and podocyte injury were worsened by GBCA. Similarly, HFD-mediated hyperlipidemia and lipid metabolites were exacerbated by gadolinium. This is the first evidence that GBCA causes significant metabolic disorders and kidney injury in mice without renal insufficiency and that the injurious actions of GBCA are amplified by obesity. The understanding of the functional interplay between gadolinium and obesity will allow the development of therapeutic interventions or the establishment of effective preventive measures to reduce gadolinium-and obesity-mediated renal pathologies.

Systemic fibrosis can be induced in humans with gadolinium-based contrast, and cumulative doses correlate with severity. Bone marrow-derived fibrocytes accumulate in the dermis. Whether target organs liberate chemokines to recruit these fibrocytes or whether fibrocytes are stimulated to home to the affected tissue is unknown. Transgenic (tagged) donor rats were treated with gadolinium-based contrast. Bone marrow was obtained from diseased animals and age-matched controls. Rats with subtotal nephrectomies were lethally irradiated and underwent salvage transplantation with either the contrast-naïve or contrast-exposed bone marrow. Groups were randomly assigned to control or contrast treatment. Contrast treatment led to dermal fibrosis, and this was exacerbated in recipients of contrast-exposed marrow. Fibronectin, C-C chemokine receptors (CCRs)2 and 7, and oxidative stress were all increased in skin from contrast-treated animals-all parameters more severe in recipients of contrast-treated animals. The respective ligands, monocyte chemoattractant protein and C-C motif ligand 19, were both elevated in skin from contrast-treated animals. Coadministration of gadolinium-based contrast and a CCR2 inhibitor reduced the severity of skin disease as well as dermal cellularity. The functional role of chemokines in the effects of gadolinium-based contrast was further confirmed in in situ coculture studies using neutralizing CCR2 antibodies. These data implicate dermal liberation of specific chemokines in the recruitment of circulating bone marrow-derived cells. The disease is augmented by bone marrow exposure to contrast, which explains why multiple exposures correlate with severity.-Drel, V. R., Tan, C., Barnes, J. L., Gorin, Y., Lee, D.-Y., Wagner, B. Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis.

Objective We previously demonstrated that NADPH oxidase 4 (Nox4) mediates increased monocyte priming and chemotaxis under conditions of diabetic metabolic stress (DMS), and emerging data indicate that Group VIA Phospholipase A2 (iPLA2β) also participates in regulating monocyte chemotaxis. Here we examined relationships between iPLA2β expression and Nox4 action in mouse peritoneal macrophages subjected to DMS. Approach and Results Increased iPLA2β expression and activity were observed in macrophages from low density lipoprotein receptor knockout (LDLR−/−) mice that were fed a high fat diet (HFD), and this was associated with time-dependent increases in atherosclerotic lesion size and macrophage content. Incubating macrophages with 30 mM D-glucose (HG), 100 μg/ml LDL, or both (HG+LDL) induced a robust increase in iPLA2β expression and activity and in cell migration in response to monocyte chemoattractant protein-1 (MCP-1). The increases in iPLA2β activity and cell migration were prevented by a bromoenol lactone (BEL) iPLA2β suicide inhibitor or an iPLA2β antisense oligonucleotide. Incubating macrophages under conditions that mimic DMS ex vivo resulted in increased Nox4 expression and activity and H2O2 generation compared to controls. BEL prevented those effects without affecting Nox2 expression. Nox4 inhibition eliminated DMS-induced acceleration of macrophage migration. Lysophosphatidic acid (LPA) restored Nox4 expression, H2O2 generation, and migration to BEL-treated cells, and an LPA receptor antagonist abrogated iPLA2β-mediated increases in Nox4 expression. Conclusions Taken together, these observations identify iPLA2β and LPA derived from its action as critical in regulating macrophage Nox4 activity and migration in the diabetic state in vivo and under similar conditions ex vivo.