Abstract-We tested whether or not complement activation participates in angiotensin (Ang) II-induced vasculopathy. We used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the human renin inhibitor aliskiren. Sprague-Dawley (SD) rats were controls. DTGR had increased blood pressure at week 5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7. C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-␣, C1q, C3, C3c, and C5b-9 expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after Ang II, TNF-␣, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-␣ and CRP induced C3 mRNA slightly in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation and cell infiltration occurred before the onset of albuminuria in Ang II-mediated renal damage. TNF-␣ and CRP played a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang II-induced model, complement activation is a major participant and suggest that TNF-␣ and CRP may play a role in its induction. Key Words: angiotensin II Ⅲ complement Ⅲ immune system Ⅲ albuminuria and renal damage T he innate complement system eliminates invading pathogens, stimulates opsonization, enhances phagocytosis, cytolysis, chemotaxis, and solubilizes immune complexes. Complement forms a bridge between innate and acquired immunity. 1,2 On excessive activation or inappropriate deposition, complement can cause disease. 3 The classical alternative and lectin complement pathway merge at the level of C3, resulting in the generation of C5b-9, the membrane attack complex. Complement activation has been implicated in the pathogenesis of numerous proteinuric renal diseases including glomerulonephritis, transplant rejection, and ischemiareperfusion injury. [3][4][5][6][7] Pratt et al demonstrated that the absence of local C3 production modulates renal graft survival and regulates T-cell priming of donor antigens. 4 Very recently, Lin et al reported that young spontaneous hypertensive rats (SHR) that have not yet developed hypertension showed increased C3 expression and increased vascular smooth muscle cell (VSMC) proliferation. Both were blocked by C3 downregulation. 8 Several studies showed that angiotensin (Ang) II not only is a vasoconstrictor peptide but also promotes inflammation and renal damage. We showed recently that immunosuppression improved nonimmune Ang II-mediated renal damage. 9 The evidence that Ang II affects the complement system is indirect. Abbate et al demons...
Abstract-Nuclear factor kappa B (NF-B) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell-specific NF-B suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell-restricted NF-B super-repressor IB␣⌬N (Tie-1-⌬N mice) overexpression. We confirmed cell-specific IB␣⌬N expression and reduced NF-B activity after TNF-␣ stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-Larginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-⌬N and control mice. In contrast to control mice, Tie-1-⌬N mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-B targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-B activation and hypertension-induced renal damage. We conclude that in vivo NF-B suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure. Key Words: hypertension Ⅲ endothelium Ⅲ NF-B Ⅲ target-organ damage H ypertension is a risk factor for target-organ damage such as cardiac and renal disease. 1 Endothelial cell injury initiates adhesion molecule and chemokine expression promoting inflammation that contributes to the pathogenesis of hypertension-induced target organ damage. [2][3][4][5] Patients with severe hypertension and target-organ damage often show elevated angiotensin (Ang) II levels and reduced nitric oxide (NO) production. Ang II signaling blockade reduces blood pressure and blunts the development and progression of vascular disease in small and large vessels in experimental animal models and in humans. Ang II also elicits an inflammatory response in both endothelial cells 6 and vascular smooth muscle cells. 7,8 Numerous in vitro and in vivo studies demonstrated that Ang II activates the nuclear factor kappa B (NF-B), a major transcription factor in mediating inflammation and innate immunity. 4,6 -8 In resting cells, NF-B resides inactive in the cytoplasm by forming complexes with the inhibitor of B (I〉) proteins. Exposure to extracellular stimuli like TNF-␣, IL-1, reactive oxygen species, Ang II, and numerous other activators leads to rapid phosphorylation, site-specific ubiquitination, and subsequent degradation of the IB proteins by the 26S proteasome. 9 The resulting free NF-B molecules translocate to the nucleus and regulate target gene expression. The targets include genes involved in the control of cell proliferation, apoptosis, innate, and adaptive immune response. 10,11 NF-B and inflammation play an important role in the development of the target organ damage. 4,[12][13][14][15][16][17] However, inhibition of NF-B signaling could also be detrimental. 18,19 Furthermore, the ideal site for NF-B inhibition in the vesse...
The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans andA. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 101 and 104 Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR–enzyme-linked immunosorbent assay analysis.
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