The reduced expression of angiotensin-converting enzyme (ACE) 2 in the kidneys of animal models and patients with diabetes suggests ACE2 involvement in diabetic nephrology. To explore the renoprotective effects of ACE2 overexpression, ACE inhibition (ACEI) or both on diabetic nephropathy and the potential mechanisms involved, 50 Wistar rats were randomly divided into a normal group that received an injection of sodium citrate buffer and a diabetic model group that received an injection of 60 mg/kg streptozotocin. Eight wks after streptozotocin injection, the diabetic rats were divided into no treatment group, adenoviral (Ad)-ACE2 group, Ad-green flurescent protein (GFP) group, ACEI group receiving benazepril and Ad-ACE2 + ACEI group. Four wks after treatment, physical, biochemical, and renal functional and morphological parameters were measured. An experiment in cultured glomerular mesangial cells was performed to examine the effects of ACE2 on cellular proliferation, oxidative stress and collagen IV synthesis. In comparison with the Ad-GFP group, the Ad-ACE2 group exhibited reduced systolic blood pressure, urinary albumin excretion, creatinine clearance, glomeruli sclerosis index and renal malondialdehyde level; downregulated transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and collagen IV protein expression; and increased renal superoxide dismutase activity. Ad-ACE2 and ACEI had similar effects, whereas combined use of Ad-ACE2 and ACEI offered no additional benefits. ACE2 transfection attenuated angiotensin (Ang) II-induced glomerular mesangial cell proliferation, oxidative stress and collagen IV protein synthesis. In conclusion, ACE2 exerts a renoprotective effect similar to that of ACEI treatment. Decreased renal Ang II, increased renal Ang-(1-7) levels, and inhibited oxidative stress were the possible mechanisms involved.

Objective-To test the hypothesis that chronic infusion of angiotensin-(1-7) [Ang-(1-7)] may dose-dependently inhibit atherosclerotic lesion formation by targeting vascular smooth muscle cells and a large dose of Ang-(1-7) may stabilize mature plaque by targeting macrophages. Approach and Results-In vivo, the effects of Ang-(1-7) on atherogenesis and plaque stability were observed in ApoE −/− mice fed a high-fat diet and chronic angiotensin II infusion. In vitro, the effects of Ang-(1-7) on vascular smooth muscle cells' proliferation and migration, and macrophage inflammatory cytokines were examined. Ang-(1-7) dosedependently attenuated early atherosclerotic lesions and inhibited vascular smooth muscle cells' proliferation and migration via suppressing extracellular regulated protein kinase/P38 mitogen-activated protein kinase and janus kinase/ signal transducers and activators of transcription activities and enhancing smooth muscle 22α and angiotensin II type 2 receptor expression. Ang-(1-7) treatment resulted in high contents of collagen and vascular smooth muscle cells, and low contents of macrophages and lipids in carotid mature plaques. Ang-(1-7) lowered the expression levels of proinflammatory cytokines and activities of matrix metalloproteinases in mature plaques. Conclusions-Ang-(1-7) treatment inhibits early atherosclerotic lesions and increases plaque stability in ApoE−/− mice, thus providing a novel and promising approach to the treatment of atherosclerosis. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Body Weight, Blood Pressure, and Serum Lipid ProfileAt the end of the first part (8 weeks) and the second part (10 weeks) of the in vivo study, both systolic and diastolic blood pressures were substantially increased in comparison with the baseline blood pressure measurements (data not shown). However, no significant differences were found in body weight, blood pressure, and serum lipid levels among vehicle-treated, Ang-(1-7)-treated and Ang-(1-7)+A779-treated groups, indicating that chronic infusion of Ang-(1-7) or A779 had no significant effects on these parameters (Table I in the online-only Data Supplement). In ApoE −/− mice without Ang-II infusion, blood pressure levels were not statistically different among vehicle-treated, Ang-(1-7)-treated, and Ang-(1-7)+A779-treated groups ( Figure 1; Table II in the online-only Data Supplement). Aortic Lesion FormationIn the first part of the in vivo study, the relative en face lesion area of the aorta arches was dose-dependently decreased in the Ang-(1-7)-treated subgroups. However, only the difference between the large dose of Ang-(1-7) subgroup and the vehicle-treated group reached a statistical significance. The antiatherosclerosis effect of Ang-(1-7) was significantly reversed by coadministration of A779 (Figure 2A and 2B). Similarly, the relative cross-sectional area of the aortic lesion also showed a dose-dependent decrease in the Ang-(1-7)-treated subgroups but only the difference between the large do...

An abundant increase in infl ammatory factors leads to acute infl ammatory diseases, such as acute coronary syndrome (ACS) ( 1 ). C-reactive protein (CRP), the prototypic marker of infl ammation, is one of the strongest predictors of cardiovascular events ( 2 ). Recent studies have demonstrated that CRP is present in atherosclerotic plaques and plays a pivotal role in promoting atherogenesis by regulating the expression and release of infl ammatory cytokines ( 3-5 ).Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), a type II membrane glycoprotein acting as a receptor for oxidized low-density lipoprotein, mediates vascular dysfunction ( 3 ). LOX-1 is expressed on the cell surface and can be proteolytically cleaved at its extracellular domain and released as a soluble form (sLOX-1) ( 6 ). sLOX-1 level refl ects increased oxidative stress of vascular walls and has been identifi ed as a novel marker for early diagnosis of ACS ( 7,8 ). However, the exact mechanisms of sLOX-1 release from the cell membrane are poorly understood.Tumor necrosis factor-a converting enzyme (TACE), a disintegrin and metalloproteinase, mediates the release of growth factors, receptors, and adhesion molecules ( 9 ). TACE is synthesized in a latent form and activated by reactive oxygen species (ROS) before reaching the cell membrane ( 10, 11 ). Studies report that CRP can upregulate ROS production by activating NAPDH Abstract Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the infl ammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-␣ converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-␣ and further treated with CRP in the absence or presence of specifi c inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dosedependent manner and that these effects were regulated by China (No. 60831003), and the National Natural Science Foundation of China (Nos. 30700301, 30971096, 30972809, and 81000126 Abbreviations: ACS, acute coronary syndrome; Apo, apocynin; CRP, C-reactive protein; DCF-DA, 2 ′ ,7 ′ -dichlorodihydrofl uorescein diacetate; Fc g R, Fc gamma receptor; IL, interleukin; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; NAC, N-acetylcysteine; PBMC, peripheral blood mononuclear cell; PMSF, phenylmethyl sulfonylfl uoride; ROS, reactive oxygen species; siRNA, small interfering RNA; TACE, tumor necrosis factor-a converting enzyme; TAPI-1, tumor necrosis factor-a protease inhibitor 1; TNF-a , tumor necrosis factor-a . 1 X. Q. Zhao and M. W. Zhang contributed equally to this work.

The noncollagenous (NC1) domains of α3α4α5(IV) collagen in the glomerular basement membrane (GBM) are targets of Goodpasture autoantibodies or Alport posttransplant nephritis alloantibodies mediating rapidly progressive glomerulonephritis. Because the autoepitopes but not the alloepitopes become cryptic upon assembly of α3α4α5NC1 hexamers, we investigated how the accessibility of B cell epitopes in vivo influences the development of glomerulonephritis in mice passively immunized with human anti-GBM Abs. Alport alloantibodies, which bound to native murine α3α4α5NC1 hexamers in vitro, deposited linearly along the mouse GBM in vivo, eliciting crescentic glomerulonephritis in Fcgr2b−/− mice susceptible to Ab-mediated inflammation. Goodpasture autoantibodies, which bound to murine α3NC1 monomer and dimer subunits but not to native α3α4α5NC1 hexamers in vitro, neither bound to the mouse GBM in vivo nor induced experimental glomerulonephritis. This was due to quinary NC1 crosslinks, recently identified as sulfilimine bonds, which comprehensively locked the cryptic Goodpasture autoepitopes in the mouse GBM. In contrast, non-crosslinked α3NC1 subunits were identified as a native target of Goodpasture autoantibodies in the GBM of squirrel monkeys, a species susceptible to Goodpasture autoantibody-mediated nephritis. Thus, crypticity of B cell autoepitopes in tissues uncouples potentially pathogenic autoantibodies from autoimmune disease. Crosslinking of α3α4α5NC1 hexamers represents a novel mechanism averting autoantibody binding and subsequent tissue injury by posttranslational modifications of an autoantigen.

In the present study, we tested our hypothesis that atorvastatin exerts its antiinflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-B signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin ϩ LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-B binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin ϩ LPSinduced NF-B activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin ϩ LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-B activity. On the other hand, blocking NF-B with SN50 produced no effects on atorvastatin ϩ LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized I-B␣, which directly inhibited NF-B activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-B activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-B.atherosclerosis; inflammation; NF-B TREATMENT WITH STATINS, competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA coenzyme A reductase, not only reduces the incidence of cardiovascular events (9) but also improves outcomes in patients with sepsis (1). Besides their cholesterollowering effect, statins have anti-inflammatory and immunomodulatory benefits. They can inhibit lipopolysaccharide (LPS)-mediated activation of human peripheral mononuclear cells and endothelial cells (18) and reduce the level of the proinflammatory cytokines tumor necrosis factor ␣ (TNF-␣) and interleukin (IL)-6 (23), thereby suppressing vascular inflammation and stabilizing vulnerable plaques (17). However, little is known about the mechanisms responsible for these anti-inflammatory effects.LPS is a unique glycolipid comprising most of the outer leaflet of the outer wall of Gram-negative bacteria, and LPS recognition and signal transmission are among the key events in the host defense reaction against Gram-negative bacteria. LPS may be linked to vascular disease, and low levels of circulating endotoxin in humans and rabbits have been shown to promote the development of atherosclerosis (11).Toll-like receptors (TLRs) are type-I transmembrane receptors expressed on the cell membrane after LPS stimulation. They are...