Regulatory T cells ameliorate angiotensin II-induced cardiac damage.

BACKGROUND: Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4+CD25+ regulatory T (Treg) cells in the pathogenesis of hypertensive target organ damage is unexplored. METHODS AND RESULTS: We conducted adoptive transfer of Treg cells into angiotensin II-infused hypertensive mice. Treg cell recipients exhibited improved cardiac hypertrophy and less cardiac fibrosis despite sustained hypertension. Amelioration of cardiac morphology was accompanied by an improvement in arrhythmogenic electric remodeling, indicating the functional significance of the enhanced cardiac morphology. Delocalization of the connexin 43 gap junction protein is one of the major pathomechanisms in electric remodeling. Pronounced connexin 43 immunoreactivity was found at the lateral borders of cardiomyocytes in angiotensin II-treated mice. In contrast, connexin 43 was restricted to the intercalated disk regions in sham controls. Surprisingly, angiotensin II+Treg-treated mice showed normal connexin 43 gap junction protein localization. Adoptive Treg cell transfer resulted in a marked reduction in cardiac CD4+, CD8+, and CD69+ cell and macrophage infiltration. CONCLUSIONS: Immunosuppressive effects of transferred Treg cells ameliorated cardiac damage and accounted for the improved electric remodeling independently of blood pressure-lowering effects. Our results provide new insights into the pathogenesis of hypertensive cardiac damage and could therefore lead to new therapeutic approaches that involve manipulation of the immune system.

Novel role for inhibitor of differentiation 2 in the genesis of angiotensin II-induced hypertension.

BACKGROUND: Angiotensin (Ang) II-induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2(-/-)) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II-induced target-organ damage. METHODS AND RESULTS: We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II-infused Id2(+/-), Id2(-/-) mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2(+/-) bone marrow to Id2(-/-) mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2(-/-) kidneys to Id2(+/-) mice also could not prevent Ang II-induced hypertension and renal damage. We verified the Ang II resistance in Id2(-/-) mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2(-/-) or Id2(+/-) mice. Angiotensinogen-transgenic Id2(+/-) mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2(-/-) mice did not. We also found that vascular smooth muscle cells from Id2(-/-) mice showed an antisenescence phenotype. CONCLUSIONS: Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II-induced hypertension.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Vascular endothelial cell-specific NF-kappaB suppression attenuates hypertension-induced renal damage.

Nuclear factor kappa B (NF-kappaB) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell-specific NF-kappaB suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell-restricted NF-kappaB super-repressor IkappaBalphaDeltaN (Tie-1-DeltaN mice) overexpression. We confirmed cell-specific IkappaBalphaDeltaN expression and reduced NF-kappaB activity after TNF-alpha stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-l-arginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-DeltaN and control mice. In contrast to control mice, Tie-1-DeltaN mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-kappaB targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-kappaB activation and hypertension-induced renal damage. We conclude that in vivo NF-kappaB suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure.

Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation.

AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage.

Complement activation in angiotensin II-induced organ damage.

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)-alpha, 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-alpha, 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-alpha 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-alpha 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-alpha and CRP may play a role in its induction.

Aldosterone synthase inhibitor ameliorates angiotensin II-induced organ damage.

BACKGROUND: Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland. METHODS AND RESULTS: We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week 7 (177+/-6 mm Hg in dTGR+FAD286 and 200+/-5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190+/-9 and 187+/-4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (P<0.05). CONCLUSIONS: Aldosterone plays a key role in the pathogenesis of Ang II-induced organ damage. Both FAD286 and ADX reduced circulating and cardiac aldosterone levels. The present results show that aldosterone produced in the adrenals is the main source of cardiac aldosterone.

The effect of fever-like temperatures on neutrophil signaling.

The effect of fever on neutrophils has not been explored. We tested the hypothesis that fever-like temperature spikes affect neutrophil signaling and function. Prior 60 min, 42 degrees C heat exposure inhibited p38 MAPK, ERK, PI3-Kinase/Akt, and NF-kappaB activation in TNF-alpha-challenged suspended neutrophils. Using pharmacological inhibitors and an inhibitory peptide transduced into neutrophils by a HIV-TAT sequence, we found that p38 MAPK and NF-kappaB mediate TNF-alpha-mediated delayed apoptosis in suspended neutrophils. Heat exposure (39-42 degrees C) did not affect constitutive apoptosis but abrogated TNF-alpha-delayed apoptosis in these suspended cells. In contrast, adhesion-dependent functions were not inhibited. Furthermore, we found that heat exposure neither blocked p38 MAPK, ERK, and NF-kappaB activation in neutrophils on fibronectin nor prevented delayed apoptosis by TNF-alpha when cells interacted with fibronectin. Above and beyond apoptosis, TNF-alpha initiated NF-kappaB-dependent gene transcription. Heat exposure blocked this effect in suspended neutrophils but not in neutrophils on fibronectin. Finally, we show that beta2-integrins, which are not necessary for TNF-alpha-induced NF-kappaB activation at 37 degrees C, transduce costimulatory signals allowing NF-kappaB activation after heat exposure. The effect could protect circulating neutrophils from TNF-alpha activation, while not interfering with activation of adherent neutrophils. Fever could make neutrophils more parsimonious.

Cardiac gene expression profile in rats with terminal heart failure and cachexia.

About one-half of double transgenic rats (dTGR) overexpressing the human renin and angiotensinogen genes die by age 7 wk of terminal heart failure (THF); the other (preterminal) one-half develop cardiac damage but survive. Our study's aim was to elucidate cardiac gene expression differences in dTGR-THF compared with dTGR showing compensated cardiac hypertrophy but not yet THF. dTGR treated with losartan (LOS) and nontransgenic rats (SD) served as controls. THF-dTGR body weight was significantly lower than for all other groups. At death, THF-dTGR had blood pressures of 228 +/- 7 mmHg (cardiac hypertrophy index 6.2 +/- 0.1 mg/g). Tissue Doppler showed reduced peak early (Ea) to late (Aa) diastolic expansion in THF-dTGR, indicating diastolic function. Preterminal dTGR had blood pressures of 197 +/- 5 mmHg (cardiac hypertrophy index 5.1 +/- 0.1 mg/g); Ea < Aa compared with LOS-dTGR (141 +/- 6 mmHg; 3.7+/-0.1 mg/g; Ea > Aa) and SD (112 +/- 4 mmHg; 3.6 +/- 0.1 mg/g; Ea > Aa). Left ventricular RNA was isolated for the Affymetrix system and TaqMan RT-PCR. THF-dTGR and dTGR showed upregulation of hypertrophy markers and alpha/beta-myosin heavy chain switch to the fetal isoform. THF-dTGR (vs. dTGR) showed upregulation of 239 and downregulation of 150 genes. Various genes of mitochodrial respiratory chain and lipid catabolism were reduced. In addition, genes encoding transcription factors (CEBP-beta, c-fos, Fra-1), coagulation, remodeling/repair components (HSP70, HSP27, heme oxygenase), immune system (complement components, IL-6), and metabolic pathway were differentially expressed. In contrast, LOS-dTGR and SD had similar expression profiles. These data demonstrate that THF-dTGR show an altered expression profile compared with preterminal dTGR.

Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25-OH vitamin D3 to 1,25-(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal-specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.

Vascular smooth muscle and nitric oxide synthase.

The concept of endothelium-derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP-dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium-independent manner.

L-Arginine-NO-cGMP signaling following acute liver injury in the rat.

The incidence of liver diseases has increased over the past few years. For this reason, the consequences of induced nitric oxide (NO) synthesis in liver damages warrant further studies. To address this issue, we investigated the expression of key enzymes engaged in the control of NO signaling in the rat liver after carbon tetrachloride (CCl4) intoxication and subsequent regeneration. CCl4 intoxication resulted in up-regulation of the entire NO signal transduction machinery. Expression patterns of arginase, soluble guanylyl cyclase and cyclic nucleotide phosphodiesterase revealed striking parallels with that of NO synthase (NOS). Co-expression of the major components of the l-arginine-NO-cGMP signaling cascade both in hepatocytes and in nonparenchymal cells indicates an autocrine rather than a paracrine fashion of NO signaling in the liver. Up-regulation of NOS after CCl4 intoxication fell behind the oxidative stress and was found to be associated with the initiation of parenchymal regeneration implying a beneficial effect of NO.

Myopathy-dependent changes in activity of ATPase, SDH and GPDH and NOS expression in the different fibre types of hamster muscles.

Proximal (vastus lateralis) and distal (gastrocnemius) muscles of 100-day-old normal and myopathic BIO TO-2 hamsters were analysed to study the effects of myopathy on the different muscle fibre types: SO (slow oxidative), FOG (fast oxidative glycolytic) and FG (fast glycolytic). Cytophotometric measurements of enzyme activities (myofibrillic adenosine triphosphatase, succinate dehydrogenase and glycerol-3-phosphate dehydrogenase), Western blot analysis of nitric oxide synthase (NOS) I, II, III isoforms and NOS II immunohistochemistry were performed. The following alterations were found in myopathic muscle fibres: all fibre types of both proximal and distal myopathic muscles showed decreased myofibrillic adenosine triphosphatase activity indicating depressed contractility. This was associated with depressed oxidative activity of the muscle fibres. A shift to more glycolytic metabolism was observed, mainly in FG fibres of proximal muscle. We found an increased NOS II expression in both myopathic muscle types investigated. It means that increased NO production inhibits force generation in myopathic muscle. NOS II immunoreactivity was found mainly in the cytoplasm of FG fibres. NOS I and NOS III expression was not significantly effected by this form of myopathy. Our findings demonstrate that muscle fibres of proximal and distal skeletal muscles of 100-day-old cardiomyopathic BIO TO-2 hamsters are altered with respect to contractility, metabolism and NOS II expression. FG fibres of the proximal muscle were effected most strongly.

Aldosterone abrogates nuclear factor kappaB-mediated tumor necrosis factor alpha production in human neutrophils via the mineralocorticoid receptor.

Mineralocorticoid receptor (MR) activation by aldosterone controls salt homeostasis and inflammation in several tissues and cell types. Whether or not a functional MR exists in polymorphonuclear neutrophils is unknown. We investigated the hypothesis that aldosterone modulates inflammatory neutrophil responses via the MR. By flow cytometry, Western blot analysis, and microscopy, we found that neutrophils possess MR. Preincubation with aldosterone (10(-11) to 10(-6) M) dose-dependently inhibited nuclear factor kappaB activation in interleukin (IL)-8- and granulocyte/macrophage colony-stimulating factor-treated neutrophils on fibronectin by IkappaBalpha Western blotting, electrophoretic mobility shift assay, and RT-PCR for IkappaBalpha mRNA. Aldosterone had no effect on tumor necrosis factor alpha- and lipopolysaccharide-mediated nuclear factor kappaB activation or on IL-8- and granulocyte/macrophage colony-stimulating factor-induced extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or phosphatidylinositol 3-kinase/Akt activation. Spironolactone prevented nuclear factor kappaB inhibition, indicating an MR-specific aldosterone effect. By RT-PCR, we found that neutrophils have 11beta-hydroxysteroid dehydrogenase. Tumor necrosis factor alpha, which is controlled by nuclear factor kappaB, increased in the cell supernatant with IL-8 treatment. Aldosterone completely prevented this effect. RT-PCR showed a strong tumor necrosis factor alpha mRNA increase with IL-8 that was blocked by aldosterone, excluding the possibility that the tumor necrosis factor alpha increase was merely a consequence of secretion. Finally, conditioned medium from IL-8-treated neutrophils increased intercellular adhesion molecule-1 expression on endothelial cells and subsequently the adhesion of IL-8-treated neutrophils to endothelial cells. These effects were reduced when conditioned medium from aldosterone-pretreated neutrophils was used, and spironolactone blocked the aldosterone effect. Our data indicate that a functional MR exists in neutrophils mediating antiinflammatory effects that are at work when neutrophils interact with endothelial cells. These data could be relevant to MR-blockade treatment protocols.

Short-term heat exposure inhibits inflammation by abrogating recruitment of and nuclear factor-{kappa}B activation in neutrophils exposed to chemotactic cytokines.

Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides beta2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-kappaB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-kappaB activation in a mouse model of skin inflammation. Mice that were exposed to 40 degrees C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-kappaB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-kappaB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IkappaBalpha mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation.

Trophoblasts reduce the vascular smooth muscle cell proatherogenic response.

Maternal spiral artery remodeling is the consequence of controlled trophoblast invasive interaction with the maternal cellular environment and is fundamentally important for successful placentation. In preeclampsia, trophoblast invasion is shallow, remodeling is incomplete, and vessels develop an inflammatory appearance, termed "acute atherosis." We noted that, in our preeclampsia, human renin-human angiotensinogen transgenic rat model, complement component 3 (C3), and tumor necrosis factor-alpha were upregulated and heavily expressed in atherotic uteroplacental vessels. We next used coculture involving human trophoblasts, rat vascular smooth muscle cells (VSMCs), and human VSMCs to observe VSMC-trophoblast regulatory interactions. Tumor necrosis factor-alpha induced complement C3 and interleukin-6 expression in VSMCs. We found that trophoblasts were able to reduce VSMC C3 and interleukin-6 expression after the VSMCs were stimulated with tumor necrosis factor-alpha. However, a direct VSMC-trophoblast cell-cell contact was necessary for this anti-inflammatory response. We also studied double-transgenic VSMCs that express inflammatory components and exhibit accelerated proliferation ("synthetic" phenotype). Trophoblasts could not downregulate C3 in these cells. We then examined uteroplacental tissues from preeclamptic and control patients. In control deciduas, only traces of C3 staining were observed, and vessels were thin walled without thrombus formation. In preeclampsia, the decidual vessels showed atherosis, thrombus formation, and C3 expression. Our data suggest that fetally derived trophoblasts require direct cell-cell contact with maternally derived VSMCs to downregulate VSMC C3 and interleukin-6 expression and to avoid atherosis. The findings also implicate C3 in the placental vasculopathy observed in preeclampsia.

Role of the multidomain protein spinophilin in blood pressure and cardiac function regulation.

Spinophilin controls intensity/duration of G protein-coupled receptor signaling and thereby influences synaptic activity. We hypothesize that spinophilin affects blood pressure through central mechanisms. We measured blood pressure and heart rate in SPL-deficient (SPL(-/-)), heterozygous SPL-deficient (SPL(+/-)), and wild-type (SPL(+/+)) mice by telemetry combined with fast Fourier transformation. We also assessed peripheral vascular reactivity and performed echocardiography. SPL(-/-) had higher mean arterial pressure than SPL(+/-) and SPL(+/+) (121+/-2, 112+/-1, and 113+/-1 mm Hg). Heart rate was inversely related to spinophilin expression (SPL(-/-) 565+/-0.4, SPL(+/-) 541+/-5, SPL(+/+) 525+/-8 bpm). The blood pressure response to prazosin, trimethapane, and the heart rate response to metoprolol were stronger in SPL(-/-) than SPL(+/+) mice, whereas heart rate response to atropine was attenuated in SPL(-/-). Mesenteric artery vasoreactivity after angiotensin II, phenylephrine, and the thromboxane mimetic (U46619) as well as change in heart rate, stroke volume, and cardiac output after dobutamine were similar in SPL(-/-) and SPL(+/+). Baroreflex sensitivity was attenuated in SPL(-/-) compared with SPL(+/-) and SPL(+/+), which was confirmed by pharmacological testing. Heart rate variability parameters were attenuated in SPL(-/-) mice. We suggest that an increase in central sympathetic outflow participates in blood pressure and heart rate increases in SPL(-/-) mice. The elevated blood pressure in SPL(-/-) mice was associated with attenuated baroreflex sensitivity and decreased parasympathetic activity. Our study is the first to show a role for the spinophilin gene in blood pressure regulation.

Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide.

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of (125)I-renin or (125)I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

Beta2-integrins and acquired glycoprotein IIb/IIIa (GPIIb/IIIa) receptors cooperate in NF-kappaB activation of human neutrophils.

Microparticles from various cells are generated during inflammation. Platelet-derived microparticles (PMPs) harbor receptors that are not genuinely expressed by neutrophils. We tested whether or not functional glycoprotein IIb/IIIa (GPIIb/IIIa) receptors can be acquired by neutrophils via PMPs and whether these receptors participate in pro-inflammatory signaling. Surface expression was analyzed by flow cytometry and confocal microscopy. NF-kappaB activation was analyzed by Western blot experiments, electrophoretic mobility shift assays, and reverse transcription-PCR. Cell adhesion and spreading were estimated by myeloperoxidase assay and light microscopy. We found that PMPs transfer GPIIb/IIIa receptors to isolated and whole blood neutrophils via PMPs. We used specific antibodies in granulocyte macrophage colony-stimulating factor-treated neutrophils and observed that acquired GPIIb/IIIa receptors co-localized with beta2-integrins and cooperated in NF-kappaB activation. We show that Src and Syk non-receptor tyrosine kinases, as well as the actin cytoskeleton, control NF-kappaB activation. In contrast to NF-kappaB, acquisition of GPIIb/IIIa receptors was not necessary to induce adhesion to fibronectin or phosphatidylinositol 3-kinase/Akt signaling. When granulocyte macrophage colony-stimulating factor-stimulated neutrophils were incubated on fibronectin, strong NF-kappaB activation was observed, but only after loading with PMPs. Blocking either beta2-integrins or GPIIb/IIIa receptors abrogated this effect. Therapeutic GPIIb/IIIa inhibitors were similarly effective. The compounds also inhibited NF-kappaB-dependent tumor necrosis factor-alpha mRNA up-regulation. The data implicate GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced inflammation.

NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.