Vascular Endothelial Cell–Specific NF-B Suppression Attenuates Hypertension-Induced Renal Damage Norbert Henke, Ruth Schmidt-Ullrich, Ralf Dechend, Joon-Keun Park, Fatimunnisa Qadri, Maren Wellner, Michael Obst, Volkmar Gross, Rainer Dietz, Friedrich C. Luft, Claus Scheidereit, Dominik N. Muller 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. (Circ Res. 2007;101:268-276.) Key Words: hypertension 䡲 endothelium 䡲 NF-B 䡲 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.2–5 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 cells6 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.4,12–17 However, inhibition of NF-B signaling could also be detrimental.18,19 Furthermore, the ideal site for NF-B inhibition in the vessel wall is unknown. To address the role of NF-B in the endothelium in hypertension-induced renal damage, we used the Cre/lox technique to generate a novel mouse model with endothelial cell–restricted expression of the human NF-B super-repressor IB␣⌬N.14,20
Materials and Methods Mice For endothelial cell–restricted super-repressor IB␣⌬N overexpression, mice carrying loxP-flanked alleles encoding for the human IB␣⌬N (loxP-⌬N) were crossed with Cre knock-in mice expressing the Cre recombinase under the control of endothelial-specific Tie-1 promoter. Tie-1-Cre mice were a gift from Dr Erika Gustafsson.21 The loxP-⌬N mice were described previously.14,20 The Tie-1-Cre knock-in mediates excision of loxP-flanked sequences in renal, cardiac, pulmonary, and aortic endothelial cells.21 The double knock-in mice were designated Tie-1-⌬N. All mice were bred on a C57Bl/6
Original received February 12, 2007; revision received June 3, 2007; accepted June 7, 2007. From the Medical Faculty of the Charite´ (N.H., R.D., M.W., R.D., F.C.L., D.N.M.), Franz Volhard Clinic, HELIOS Klinikum-Berlin; the Max-Delbru¨ck-Center for Molecular Medicine (R.S.-U., F.Q., M.O., V.G., F.C.L., C.S., D.N.M.), Berlin-Buch; and the Medical School of Hannover (J.-K.P.), Hannover, Germany. Correspondence to Ruth Schmidt-Ullrich, PhD, Max-Delbru¨ck-Center, Robert-Rossle Street 50, 13125 Berlin-Buch, Germany. E-mail
[email protected] © 2007 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.150474
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background for more than 7 generations. The Berlin Animal Review Board approved all protocols (Reg. 0135/01 and G 0261/02).
Ang II/L-NAME Mouse Model Eight-week-old control mice (single transgenic Tie-1-Cre and loxP⌬N, wild-type C57BL/6 mice) and Tie-1-⌬N double transgenic mice (nⱖ21 for each group) received the NO synthase inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME). L-NAME was given at 0.1 mg䡠mL⫺1 in the drinking water. Ang II (1.0 mg䡠kg⫺1䡠day⫺1) was infused via ALZET mini pumps. The animals were fed a high-sodium diet (1% sodium in the chow) for 14 days.22 Because C57BL/6, Tie-1-Cre, and loxP-⌬N mice showed no differences in renal histology, albuminuria, cell infiltration, and gene expression, these groups were combined and termed controls. We measured 24-hour mouse urinary albumin by ELISA (CellTrend, Germany). Animals were killed on day 14 under ether anesthesia. Hearts and kidneys were washed, weighed, harvested, and stored until analysis.
Telemetric Mean Arterial Pressure Measurements Telemetry system and Dataquest ART 2.0 software from Data Sciences International were used to measure intracarotid blood pressure in 4 to 5 mice per group. Briefly, a telemetry probe catheter (TA11-PA40; Data Sciences International) was inserted into the surgically exposed carotid artery.23 After surgery, all mice were allowed at least 7 days to recover. Before Ang II/L-NAME treatment, baseline values were continuously recorded for 3 days and averaged. Days 10 to 12 after Ang II/L-NAME treatment, blood pressure and heart rate were recorded and statistically analyzed. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate were sampled at 100 Hz. Mean arterial pressure (MAP) was calculated as follows: MAP⫽DBP⫹(SBP-DBP)/3.
Immunohistochemistry and In Situ Hybridization Ice-cold acetone-fixed cryosections (6 m) were stained by indirect immunofluorescence techniques as described earlier.17 The sections were incubated with the following antibodies: anti-F4/80, anti-CD4 (both serotec, UK), anti-CD3, anti-CD8 (both BD Bioscience, Germany), anti-collagen III (Fitzgerald, Mass), anti-VCAM-1 (SantaCruz, Germany), anti-ICAM-1 (Dianova, Germany), anti-p65 (Abcam, UK), and anti-TNF-␣ (SantaCruz, Germany). For histology and in situ hybridization, paraffin sections were used. Masson trichrome stain was performed by clinical routine protocol. Semiquantification of tubular damage was performed as described in online methods. P65 semiquantification was evaluated with a scoring system1–5 based on the number of p65⫹ nuclei. Paraffin sections were also stained with sirius red to evaluate the degree of fibrosis. In situ hybridization was performed as described earlier.24 The murine IB␣ (nt 1 to 1091, U36277/NM010907) cDNA was used. Sections were counterstained with 0.01% PyroninG and mounted with Entellan (Merck). All pictures were taken with a Zeiss Axioplan 2 Imaging microscope/ Axiophot camera.
Endothelial Cell Culture Murine lung and aortic endothelial cells were isolated with Dynabeads coated with anti–platelet-endothelial cell adhesion molecule-1 or anti–ICAM-2 monoclonal antibody as described previously.25,26 Endothelial cells were cultured with DMEM supplemented with 20% FBS, heparin, endothelial cell growth factor, nonessential amino acids, and antibiotics.
Immunoblotting and Electrophoretic Mobility Shift Assay Endothelial cells were stimulated with TNF-␣ (20 ng/mL; Biomol, Germany) as indicated. Whole cell extracts were prepared as described earlier.27 electrophoretic mobility shift assay (EMSA) was performed with 5 g whole cell protein as described previously.27 For immunoblotting, 20 g whole cell lysate protein was used and revealed with an IB␣ antibody (Santa Cruz, Germany).
Figure 1. Generation of double transgenic Tie-1-⌬N mice is shown. The cDNA of the human IB␣ super-repressor IB␣⌬N is preceded by a stop codon flanked by 2 loxP sites. The floxed IB␣⌬N construct was integrated in frame into the -catenin locus replacing exons 3 to 6.
Quantitative TaqMan RT-PCR Total kidney RNA was isolated using the TRIZOL protocol; RNA from cells with the RNeasy kit (Qiagen, Germany). TaqMan RTPCR by SYBR Green analysis was performed as described earlier.28 ICAM-1 and VCAM-1 mRNA quantities were analyzed in duplicate. For quantification, the target sequences were normalized in relation to the mouse GAPDH product (Clonetech, Germany). Biotez (Berlin, Germany) synthesized the primers. The sequences are listed in the online method section.
Statistical Analysis Differences between experimental groups were analyzed by use of adjusted student t test or ANOVA for repeated measures, as appropriate. Data are reported as mean⫾SEM. Values of P⬍0.05 were considered significant.
Results Generation of Mice with Endothelial-Specific NF-B Inhibition
The breeding scheme of floxed IB␣⌬N (loxP-⌬N) mice and heterozygous Tie-1–Cre knock-in mice to generate mice with endothelial cell–restricted IB␣⌬N expression, namely Tie1-⌬N mice, is given (Figure 1). IB␣⌬N lacks the destruction box containing the phosphorylation and ubiquitination sites and acts as a super-repressor of NF-B activity (supplemental Figure I, available online at http://circres.ahajournals.org). To confirm the correct functioning of the Tie-1–Cre line, Tie-1– Cre mice were mated with ROSA26Rflox/flox reporter mice and endothelial cell–specific LacZ staining pattern in the aorta was observed (data not shown). These results agree with the previous description of the Tie-1-directed Cre expression.21,29 Recombination in Tie-1-⌬N mice was verified by immunoblot analysis of whole cell extracts from isolated primary endothelial cells (Figure 2A). IB␣⌬N lacks the N-terminal 70 amino acids and thus runs at a smaller molecular weight
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Figure 2. Reduced NF-B activity in primary aortic endothelial cells of Tie-1-⌬N mice. A, Whole cell extracts of unstimulated or TNF␣-stimulated primary endothelial cells isolated from aortas from Tie-1-⌬N or C57BL/6 control mice were evaluated in Western blotting. The human IB␣⌬N protein is readily detectable in the Tie-1-⌬N endothelial cells. B, EMSA analysis of the same endothelial cell extracts as in A. After TNF␣ stimulation endothelial cells of Tie-1-⌬N mice show a reduced NF-B DNA binding activity compared with wild-type controls. Extracts treated with antibodies against p50 or p65, as indicated, reveal a complete upward shift, indicating that the NF-B DNA binding complex is composed of p50/p65 heterodimers. C, Quantification of EMSA results. Results are mean⫾SEM; n⫽4; *P⬍0.05 vs TNF-␣ treated control cells. D, In situ hybridization using a DIG-labeled murine IB␣ antisense RNA probe. Strongly increased endogenous IB␣ mRNA expression in kidney sections of Ang II/L-NAME–treated control mice (blue color) is indicative for high NF-B activity in these areas. Ang II/L-NAME–treated Tie-1-⌬N revealed considerably lower IB␣ expression in vessels, tubules, and glomeruli.
range than endogenous IB␣ (Figure 2A). The efficiency of NF-B inhibition in Tie-1-⌬N was confirmed by EMSA (Figure 2B and 2C). After TNF-␣ stimulation, cultured endothelial cells from Tie-1-⌬N showed a reduced NF-B DNAbinding activity, compared with wild-type control cells (Figure 2C). Residual induced NF-B activity was most likely derived from nonrecombinant endothelial cells, because the Cre recombinase does not function 100%. Excision of loxP sites by Tie-1 promoter-induced Cre is estimated to take place in 70 and 95% of endothelial cells depending on the organ.21 Cultured wild-type endothelial cells may grow at a faster rate and eventually outnumber Tie-1-⌬N endothelial cells. Interestingly, in situ hybridization (Figure 2D) using a murine anti-sense RNA probe of the bona fide NF-B target gene IB␣ clearly demonstrated that hypertensive control mice with severe renal damage showed strong IB␣ signals in various substructures of the kidney,
namely blood vessels, tubules, and glomeruli. The increased IB␣ expression at these sites can be used as a direct read-out for NF-B activity. As expected, in all these structures we observed significantly less upregulation of IB␣ expression in Tie-1-⌬N mice and in sham-controls (Figure 2D). This result already suggests that NF-B activity in endothelial cells has a direct effect on NF-B activation in the surrounding structures such as tubules and glomeruli. We analyzed nuclear p65 immunoreactivity as a marker for NF-B activation. Vascular endothelial cells from Ang II/L-NAME-treated control mice often showed p65⫹ nuclei, whereas only a few endothelial cells were positive in from Ang II/L-NAME–treated Tie-1-⌬N mice (supplemental Figure IIA). Tubular p65 nuclei were significantly less frequent in mice with suppressed NF-B signaling compared with Ang II/L-NAME–treated control mice, but still (supplemental Figure IIB and IIC).
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Figure 3. Reduced endothelial NF-B activity has no effect on blood pressure but reduces albuminuria. A, Telemetric blood pressure recordings of control and Tie-1-⌬N mice. Tie-1-⌬N and control mice are normotensive before Ang II/L-NAME treatment (baseline) but severely hypertensive 14 days after chronic Ang II/L-NAME administration. B, Baseline urinary albumin levels before (baseline) and after 14 days of Ang II/L-NAME treatment. Albuminuria is significantly higher in controls compared with Tie-1-⌬N mice. Results are mean⫾SEM. *P⬍0.05 vs baseline; #P⬍0.05 vs Ang II/L-NAME–treated controls.
Figure 4. Hypertension-induced kidney damage is diminished in Tie-1-⌬N mice. A, Masson trichrome stain of kidney paraffin sections from Ang II/L-NAME–treated control and Tie-1-⌬N mice and from untreated controls (sham). Treated control mice have severe tubular damage with tubular necrosis (black arrows), and partial occlusion of the tubular lumen by cellular debris (black frame), but also vascular damage with increased perivascular fibrosis. In treated Tie-1-⌬N mice these histological changes are less severe. (T indicates tubules; V, vein; A, artery; G, glomerulus; yellow bar represents 200 m.) B, Semiquantification of tubular damage is shown. Results are mean⫾SEM; n⫽6 to 8; *P⬍0.05 vs Ang II/L-NAME–treated controls, #P⬍0.05 vs Ang II/L-NAME–treated Tie-1-⌬N mice. C, Sirius red stains for evaluation of renal fibrosis.
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Figure 5. Renal TNF-␣ production is greatly suppressed in hypertensive kidneys of Tie-1-⌬N mice. Cryosections of kidney tissue from Ang II/LNAME–treated control and Tie-1-⌬N mice and from sham control mice were stained with an anti– TNF-␣ antibody (in red). High TNF␣ expression is seen in damaged tubules (T), glomeruli (G), and vessels (A) of kidneys of treated control mice. Tie-1-⌬N mice show reduced amounts of TNF-␣ protein, whereas in sham-controls antibody staining is absent. (Upper panel: tubules and glomerulus; lower panel: vessel. Yellow bar represents 50 m).
NF-B Activation Promotes Hypertension-Induced Renal Damage Blood pressure recordings revealed that control mice and Tie-1-⌬N mice were normotensive before Ang II/L-NAME treatment (106⫾1 mm Hg versus 103⫾1 mm Hg, respectively; Figure 3A). Chronic Ang II/L-NAME administration raised the mean arterial blood pressure (MAP) by 40 mm Hg. There was no difference in MAP between both groups (143⫾5 mm Hg for control and 143⫾4 mm Hg for Tie-1-⌬N; Figure 3A). Baseline urinary albumin excretion was also similar between both groups (Figure 3B). However, after Ang II/L-NAME treatment, albuminuria was significantly higher in control mice compared with Tie-1-⌬N mice (6876⫾1058 versus 3022⫾737 g/d, respectively, P⬍0.05; Figure 3B). Masson trichrome stain of kidney tissue showed that Ang II/L-NAME–treated control mice had severe tubular and vascular damage with increased matrix production, compared with control mice (Figure 4A). Semiquantification of tubular damage supported this result (Figure 4B). Ang II/L-NAME– treated Tie-1-⌬N mice also showed histological changes. Nevertheless, matrix formation and tubular damage were significantly less pronounced in mice with suppressed NF-B signaling compared with Ang II/L-NAME–treated control mice (Figure 4A and 4B). Sirius red stains confirmed a significant reduction of perivascular and interstitial fibrosis in Ang II/L-NAME–treated Tie-1-⌬N mice (Figure 4C). Vascular damage was less pronounced in Ang II/L-NAME– treated Tie-1-⌬N compared with control mice (supplemental Figure IIIA). Suppressed NF-B activity only protected glomeruli to a minor degree. Glomerular collagen III deposition was partially reduced in Ang II/L-NAME–treated Tie-1-⌬N mice (supplemental Figure IIIB).
NF-B Drives Renal TNF-␣, VCAM-1, and ICAM-1 Expression in Hypertensive Mice
TNF-␣ expression was increased in damaged glomeruli, tubules, and the vessel wall from Ang II/L-NAME–treated
controls, significantly reduced in Ang II/L-NAME–treated Tie1-⌬N mice, and hardly or not expressed in sham controls (Figure 5). RT-PCR analysis revealed that after Ang II/LNAME treatment renal VCAM-1 (Figure 6A) and ICAM-1 (Figure 6B) expression was significantly increased in control mice, compared with Tie-1-⌬N mice. Nevertheless, both groups showed significantly increased adhesion molecule expression, compared with untreated normotensive shamcontrol mice (Figure 6A and 6B). Immunohistological analysis of renal VCAM-1 (Figure 6C) and ICAM-1 (Figure 6D) confirmed our RT-PCR results. Interestingly, we observed different expression patterns of VCAM-1 and ICAM-1 immunoreactivity in large or medium arteries, small arterioles, and capillaries. Large and medium arteries, as well as capillaries of treated Tie-1-⌬N mice showed a significantly reduced adhesion molecule expression compared with controls. However, arterioles showed the same staining intensity as treated controls (data not shown). This result suggests insufficient Cre recombination in arterioles. ICAM-1 was also expressed constitutively in the interstitium. However, Ang II/L-NAME–treated Tie-1-⌬N mice showed a significantly reduced renal ICAM-1 immunoreactivity in the interstitium, glomerular bed (Figure 6D upper panel), and the vessel wall (Figure 6D lower panel) compared with Ang II/L-NAME–treated controls.
Renal Inflammatory Cell Infiltration Is Controlled by NF-B Because of the increased VCAM-1 and ICAM-1 expression in Ang II/L-NAME-treated animals, we analyzed immune cell infiltration in our treated mouse models. Using an anti-CD3 antibody, elevated T-cell infiltration is seen in kidneys of treated control animals and to a lesser degree in Tie-1-⌬N animals (Figure 7A). The 2 T cell subpopulations that included CD4⫹ (helper) and CD8⫹ (cytotoxic) T-cells, as
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Figure 6. Suppressed NF-B activity leads to reduced renal VCAM-1 and ICAM-1 expression after Ang-II–LNAME treatment. Kidneys from treated and untreated animals were analyzed by real time RT-PCR for VCAM-1 (A) and ICAM-1 (B) expression. Results are mean⫾SEM; n⫽6 to 8; *P⬍0.05 vs Ang II/L-NAME– treated controls, #P⬍0.05 vs Ang II/L-NAME–treated Tie-1-⌬N mice. C, VCAM-1 immunoreactivity (red) is higher in the vascular endothelium of treated control immunohistochemistry on kidney cryosections from Ang II/L-NAME–treated control and Tie-1-⌬N mice and sham-controls. VCAM-1 kidneys, compared with Tie1-⌬N and sham kidneys. D, ICAM-1 immunofluorescence staining (red) is detected in glomeruli (G) and interstitium in kidneys of control-treated mice (upper panel). A reduced signal is seen in Tie-1-⌬N and sham kidneys (upper panel). Vascular ICAM-1 (A, artery) is shown in the lower panel. (Yellow bar represents 50 m.)
well as macrophages (F4/80⫹ cells), were primarily located around blood vessels and to a lesser extent within the interstitium. We quantified infiltrating monocytes/macrophages, T cells (CD3⫹, CD4⫹), and CD8⫹ T-cell subpopulations in stained renal sections (Figure 7B through 7E). We observed partially diminished renal immune cell infiltration in Ang II/L-NAME–treated-Tie-1-⌬N compared with Ang II/L-NAME–treated control mice (Figure 7B through 7E). Nevertheless, cell infiltration was still significantly increased in treated Tie-1-⌬N mice, compared with untreated shamcontrols (Figure 7B through 7E).
Discussion
The surprising finding is that endothelial-specific NF-B suppression suffices to ameliorate Ang II/L-NAME hypertension-induced renal damage. The results demonstrate that inhibition of NF-B signaling in a single cell type is able to reduce renal tubular, vascular, and to a minor extent also glomerular damage. Endothelial NF-B inhibition did not affect the development of hypertension, indicating that the renal protection was blood pressure– independent. Our study provides the first in vivo evidence that specific endothelial NF-B inhibition results in a
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Figure 7. Decreased cell infiltration in hypertensive kidney tissue of Tie-1-⌬N mice. A, A representative photomicrograph of T-cell infiltration illustrated by anti-CD3 antibody staining (in red) of kidney sections from Ang II/L-NAME–treated controls, Tie-1-⌬N mice, and sham-controls. B, Monocyte/macrophage (F4/80⫹) infiltration in Ang II--L-NAME–treated control and Tie-1-⌬N mice and in sham controls. C through E, T-cell infiltration: T cells (CD3), CD4⫹ helper T, and CD8⫹ cytotoxic T. Cells were counted per view field. Results are mean⫾SEM; n⫽6 to 8; *P⬍0.05 vs Ang II/LNAME–treated controls, #P⬍0.05 vs Ang II/LNAME–treated Tie-1-⌬N mice.
diminished upregulation of several proinflammatory NF-B target genes including VCAM-1 and ICAM-1 and reduces renal inflammation. Hypertension is a major risk factor for cardiovascular events. Nevertheless, high blood pressure is not a unique factor in the pathogenesis of target organ damage. We reported earlier that dexamethasone inhibited NF-B activity and prevented renal damage in an Ang II– dependent rat model despite blood pressures exceeding 200 mm Hg.30 In the present study, Ang II/L-NAME–treated controls and Tie-1-⌬N mice were severely hypertensive and both developed renal damage. Nevertheless, endothelial-specific NF-B suppression protected Tie-1-⌬N mice to a significant extent. NF-B plays an important role in the pathogenesis of cardiovascular disease. Cardiac NF-B inhibition by a decoy technique reduced the extent of myocardial infarction after reperfusion.31 In renal allografts, pretreatment of donor kidneys with NF-B decoys led to reduced NF-B activity and expression of VCAM-1 leading to reduced cell infiltration.32 Boyle et al showed that treatment with pyrrolidine dithiocarbamate (PDTC), a compound which acts as an antioxidant
and prevents ubiquitination and degradation of the NF-B inhibitor IB␣, blocked endothelial cell E-selectin expression in vitro and reduced cell infiltration in LPS-infused rabbits.33 We reported earlier that NF-B inhibition by PDTC ameliorated Ang II–induced cardiac hypertrophy and renal and vascular damage.4 In fact, PDTC probably not only blocks NF-B activation, because it also inhibits the ubiquitin ligase -TrCP, which is involved in other signaling pathways.34 Therefore, antioxidative, NF-B, or other signaling pathways might have accounted for the effect in vivo. New strategies with cell-type specific NF-B inhibition as described in this and other recent articles can circumvent this problem. Freund et al14 were the first to show that in mice with cardiomyocyte-specific NF-B suppression, cardiac hypertrophy was significantly reduced after Ang II treatment. Herrmann et al15 used a mouse stroke model and showed that suppressed IB kinase (IKK)-2 activity in neurons markedly reduced cerebral infarct size. The animal models described above consider NF-B as a detrimental factor in the pathogenesis of cardiovascular disease. NF-B also regulates cell survival and plays a fundamental role in normal cell physiology. Thus, NF-B inhibition may
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Henke et al also cause harmful effects. For instance, NF-B inhibition in macrophages increased atherosclerosis in LDL receptor– deficient mice.18 Furthermore, deletion of the IKK-NF-B pathway components results in massive hepatic apoptosis, defects in hematopoiesis, and death in utero.35–37 A transgenic mouse model similar to the Tie-1-⌬N mice was recently established by Kisseleva et al and differed from ours in the overexpression of a mutated IB␣ under the control of the Tie-2 promoter.19 In these mice, endothelial cell NF-B inhibition increased the sensitivity to LPS-induced toxemia and altered vascular integrity. Whether or not the 2 models have an identical transmission of the modified IB␣ in the same cell type is difficult to determine without a direct comparison; such a comparison would be interesting and perhaps revealing. One challenging goal for the future will be the elucidation of protective versus harmful effects of NF-B in cardiovascular disease. These effects seem to be cell- and time-dependent. More detailed studies are needed to investigate this important controversy. In contrast to atherosclerosis, hypertension-induced vascular injury is not generally considered as an inflammatory and immune disorder.38 We observed increased VCAM-1 and ICAM-1 expression on the endothelium with cell infiltration in hearts and kidneys of hypertensive double-transgenic rats with high Ang II. Direct renin inhibition, Ang II receptor and endothelin receptor blockade, aspirin, or dexamethasone treatment all reduced NF-B activation and ameliorated inflammatory target organ damage in that rat model.17,30,39,40 Our earlier studies and the data presented here support the efficacy of antiinflammatory approaches to cardiovascular diseases. In our Tie-1-⌬N mice, NF-B inhibition in a single cell type, namely endothelial cells, promoted renal protection. As a consequence of suppressed endothelial NF-B activity, reduced VCAM-1 and ICAM-1 expression led to strongly decreased immune cell infiltration in the surrounding tissues. These infiltrating inflammatory cells synthesize and secrete various cytokines that could contribute locally to organ damage. We focused our attention on the endothelium because this cell layer has been particularly implicated in target-organ damage through endothelial dysfunction mediated by nitric oxide synthase dysfunction, Ang II, and aldosterone.41 We purposely selected a model that we were sure would cause sufficient target-organ damage in mice.22 Our model with high Ang II and low nitric oxide availability mimics a pattern seen in patients with heart failure, diabetes mellitus, and arteriosclerosis, as reviewed elsewhere.42 Nevertheless, our study should not be generalized to situations in which nitric oxide synthase is still active. A better understanding of the complexity of NF-B in the regulation of beneficial and detrimental effects is needed. Therefore, uniquely targeted models such as the one reported here can be extended to other cell types. The approach could give new insights into both pathogenesis and novel therapeutic options.
Acknowledgments We thank Petra Berkefeld, Jana Czychi, Karin Ganzel, Juliette Bergemann, May-Britt Ko¨hler, Gabriele N’diaye, Mathilde Schmidt, and Sarah Ugowski for their excellent technical assistance.
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Sources of Funding The studies were supported by a grant-in-aid from the European Union (EuReGene). The Deutsche Forschungsgemeinschaft (DFG) supported D.N.M. and F.C.L, and D.N.M. is a Helmholtz fellow.
Disclosures None.
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Henke et al. Vascular endothelial cell-specific NF-κB suppression attenuates…
Expanded Methods Semi-quantification of tubular damge Semiquantitative scoring of tubular damage was performed by quantification of Masson Tri-chrome stains (n=5 mice per group) with the following categories: 5+:
severe tubular damage with tubular necrosis in >90% of the tubules including the
presence of partial occlusion of the tubular lumen by cellular debris 4+:
severe tubular damage with tubular necrosis in 75-90% of the tubules including the
presence of partial occlusion of the tubular lumen by cellular debris 3+:
severe tubular damage with tubular necrosis in 50-75% of the tubules without the
presence of partial occlusion of the tubular lumen by cellular debris 2+:
tubular damage with tubular necrosis in 25-50% of the tubules
1+:
tubular damage with tubular necrosis in
