[Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts]. / Determinación del efecto cicatrizante de Piper aduncum (matico) en fibroblastos humanos.

OBJECTIVES.: To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). MATERIALS AND METHODS.: After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. RESULTS.: Against the hDFa cell line, the extract had an IC50 of 200 µg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. CONCLUSIONS.: The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

Determinación del efecto cicatrizante de Piper aduncum (matico) en fibroblastos humanos / Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts

RESUMEN Objetivos. Evaluar el efecto cicatrizante del extracto hidroetanólico de Piper aduncum, en una línea celular de fibroblastos Dermales Adultos Humanos (hDFa). Materiales y métodos. El extracto se obtuvo mediante extracción sólido-líquido, fue concentrado y liofilizado. Se purificaron las proteínas del extracto mediante cromatografía líquida de alta eficacia de fase reversa (RP-HPLC); las proteínas fueron identificadas por espectrometría de masas en tándem de péptidos trípticos y se analizaron por MALDI-TOF-TOF en un espectrómetro de masa ABSciex4800. Los valores de concentración efectiva media (EC50), concentración inhibitoria media (IC50), y el porcentaje de proliferación celular; fueron determinados por ensayos con sales de tetrazolio (MTT) . La migración celular se evaluó mediante la "técnica de rayado" . Se analizó la expresión de factores de crecimiento mediante el ensayo de reacción en cadena de la polimerasa con transcriptasa reversa a tiempo real (RT- qPCR). Resultados. La línea hDFa evidenció un IC50 de 200 µg/mL con el extracto, el valor de EC50 fue 103,5 µg/mL. En el ensayo de proliferación, la proteína K2; mostró mayor actividad en la proliferación respecto de otros tratamientos (1 µg/mL). En el ensayo de migración de fibroblastos, la proteína K2 mostró mayor actividad (50 µg/mL). La expresión relativa del factor de crecimiento derivado de plaquetas (PDGF) se incrementó 8,6 veces respecto al control, en presencia de la proteína K2. Conclusiones. El extracto hidroetanólico, de Piper aduncum, así como las proteínas que contiene, incrementaron la proliferación y migración de fibroblastos dermales humanos (hDFa); así mismo, aumentaron la expresión de factores de crecimiento que intervienen en el proceso de cicatrización.

ABSTRACT Objectives. To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). Materials and Methods. After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Results. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. Conclusions. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

PIK3CA mutations in Peruvian patients with HER2-amplified and triple negative non-metastatic breast cancers.

PURPOSE: To determine the frequency of PIK3CA mutations in a Peruvian cohort with HER2-amplified and triple negative breast cancers (TNBC). METHODS: We analyzed two cohorts of 134 primary non-metastatic breast cancer patients from Peru. Cohorts consisted of 51 hormone receptors (+)/HER2-amplified breast tumor patients surgically resected as first treatment included in the ALTTO trial (ALTTO cohort) and 81 TNBC patients with residual disease after neoadjuvant treatment (neoadjuvant cohort). Genomic DNA was extracted from paraffin-embedded tumor samples. Samples from the ALTTO and neoadjuvant cohorts were taken at biopsies and from residual tumors, respectively. PIK3CA mutations were detected by sequencing DNA fragments obtained by PCR amplification of exons and their flanking introns. All of the detected PIK3CA mutations were confirmed in a second independent run of sample testing. RESULTS: PIK3CA mutations were present in 21/134 cases (15.7%). Mutations in exon 9 and 20 were present in 10/134 (7.5%) and 11/134 (8.2%), respectively. No cases had mutations in both exons. Mutations in exon 9 consisted of E545A (seven cases), E545K (two cases) and E545Q (one case); while in exon 20, mutations consisted of H1047R (10 cases) and H1047L (one case). Compared to TNBC patients, HER2-amplified patients were more likely to have PIK3CA mutated (23% vs 9.6%; P=0.034). There were no associations between mutational status of PIK3CA with estrogen receptor status (P=0.731), progesterone receptor status (P=0.921), age (P=0.646), nodal status (P=0.240) or histological grade (P=1.00). No significant associations were found between PIK3CA mutational status and clinicopathological features. CONCLUSIONS: We found a similar frequency of PIK3CA mutations to that reported in other series. Although we did not include HR+/HER2 patients, those with HER2-amplified tumors were more likely to present PIK3CA mutations compared to patients with triple negative tumors.

[Gastrointestinal Epstein Barr virus positive diffuse large B cell lymphoma in elderly: report of five cases and review of literature]. / Linfoma de celulas grandes B difuso gastrointestinal del anciano asociado al virus Epstein Barr: reporte de cinco casos y revision de la literatura.

EBV-positive diffuse large B-cell lymphoma (DLBCL) in elderly is a new entity included provisionally in the most recent WHO Classification of lymphoid neoplasms. It usually affects elderly patients and has a poor survival. The goal of this report was to evaluate clinical, endoscopic characteristics and survival of five patients with this entity and gastrointestinal afectation. From five cases, three cases had gastric infiltration, one ileon and one in cecum.

EBV-positive diffuse large B-cell lymphoma of the elderly: a case series from Peru.

EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly is an entity recently included in the WHO classification of lymphoid tumors. We have reviewed our experience and clinical outcomes of this distinct subtype of DLBCL. Between 2002 and 2009, cases of DLBCL were identified from medical records of the Hospital Nacional Edgardo Rebagliati Martins in Lima, Peru, and underwent pathological evaluation including immunohistochemistry for CD20, CD10, bcl-6, MUM1/IRF4, and EBV-encoded RNA in situ hybridization. Clinical data were gathered, tabulated, and reported descriptively. Survival analyses were performed using Kaplan-Meier estimates. Out of 199 cases of DLBCL, 28 cases of EBV-positive DLBCL of the elderly were identified. The median age was 75 years with male predominance (1.5:1). B-symptoms were present in 43%, advanced stage in 50% and International Prognostic Index (IPI) score > 2 in 57% of patients; 68% of patients had a nongerminal center (NGC) phenotype. The complete response rates to R-CHOP and CHOP were 63% and 33%, respectively. The median overall survival (OS) for the group was 5 months. In the univariate analysis, age ≥70 years, lymphocyte count <1.0 × 10(9) /L, and advanced clinical stage were associated with worse OS in patients treated with chemotherapy with and without rituximab. EBV-positive DLBCL of the elderly is a clinically aggressive entity with a short OS and typically presents with advanced stage, high IPI score, and a NGC phenotype. Further studies are needed to investigate if rituximab-containing regimens are associated with better response and OS rates in EBV-positive DLBCL of the elderly.

Linfoma de Celulas Grandes B Difuso Gastrointestinal del Anciano Asociado al Virus Epstein Barr: Reporte de Cinco Casos y Revision de la Literatura / Gastrointestinal Epstein Barr Virus positive diffuse large B cell lymphoma in elderly: report of five cases and review of literatura

El Linfoma de Células Grandes B difuso del anciano asociado al Epstein Barr es una nueva entidad incluida provisionalmente en la más reciente clasificación de neoplasias linfoides de la WHO. Usualmente afecta ancianos y tiene pobre sobrevida. El objetivo de este reporte fue evaluar las característica clínicas, endoscópicas y sobrevida de cinco pacientes portadores de esta entidad y compromiso gastrointestinal. Tres casos tuvieron infiltración gástrica y dos casos tuvieron compromiso del ileón y el ciego.

EBV-positive diffuse large B-cell lymphoma (DLBCL) in elderly is a new entity included provisionally in the most recent WHO Classification of lymphoid neoplasms. It usually affects elderly patients and has a poor survival. The goal of this report was to evaluate clinical, endoscopic characteristics and survival of five patients with this entity and gastrointestinal afectation. From five cases, three cases had gastric infiltration, one ileon and one in cecum.

Deficiency of antigen-presenting cell invariant chain reduces atherosclerosis in mice.

BACKGROUND: Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis. METHODS AND RESULTS: In low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, CD74 deficiency (Ldlr(-/-)Cd74(-/-)) significantly reduced atherosclerosis and CD25(+)-activated T cells in the atheromata. Although Ldlr(-/-)Cd74(-/-) mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr(-/-)Cd74(-/-) mice showed higher levels of anti-MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr(-/-)Cd74(-/-) mice had lower levels of all anti-MDA-LDL Ig isotypes compared with Ldlr(-/-) mice. As anticipated, only Ldlr(-/-) splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr(-/-) mice, but Ldlr(-/-) Cd74(-/-) mice remained protected. Compared with Ldlr(-/-) mice, Ldlr(-/-)Cd74(-/-) mice had higher anti-MDA-LDL autoantibody titers, fewer lesion CD25(+)-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti-heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL- or heat shock protein-65-immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr(-/-)Cd74(-/-) mice compared with Ldlr(-/-) mice. CONCLUSIONS: Invariant chain deficiency in Ldlr(-/-) mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes.

Cystatin C deficiency promotes inflammation in angiotensin II-induced abdominal aortic aneurisms in atherosclerotic mice.

An imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC(-/-)ApoE(-/-)) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II-induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC(-/-) ApoE(-/-) lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 +/- 1.4% versus 11.2 +/- 1.4%; P = 0.0003) and number of the CD4(+) T cells (ninefold; P = 0.048), increased significantly in CystC(-/-)ApoE(-/-) lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 gamma2 peptide production, which may account for increased microvascularization in CystC(-/-)ApoE(-/-) compared with ApoE(-/-) lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation.

CC chemokine receptor-1 activates intimal smooth muscle-like cells in graft arterial disease.

BACKGROUND: Graft arterial disease (GAD) limits long-term solid-organ allograft survival. The thickened intima in GAD contains smooth muscle-like cells (SMLCs), leukocytes, and extracellular matrix. The intimal SMLCs in mouse GAD lesions differ from medial smooth muscle cells in their function and phenotype. Although intimal SMLCs may originate by migration and modulation of donor medial cells or by recruitment of host-derived precursors, the mechanisms that underlie their localization within grafts and the factors that drive these processes remain unclear. METHODS AND RESULTS: This study of aortic transplantation in mice demonstrated an important function for chemokines beyond their traditional role in leukocyte recruitment and activation. Intimal SMLCs, but not medial smooth muscle cells, express functional CC chemokine receptor-1 (CCR1) and respond to RANTES by increased migration and proliferation. Although RANTES infusion in vivo promoted inflammatory cell accumulation in the adventitia of aortic allografts of wild-type and CCR1-deficient recipients, it increased GAD intimal thickening with SMLC proliferation in only the wild-type hosts. Aortic allografts transplanted into CCR1-deficient mice after wild-type bone marrow transplantation did not develop intimal lesions, which indicates that CCR1-bearing inflammatory cells do not contribute to intimal lesion formation. Moreover, RANTES induced SMLC proliferation in vitro but did not promote medial smooth muscle cell growth. Blockade of CCR5 attenuated RANTES-induced T-cell and monocyte/macrophage proliferation but did not affect RANTES-induced SMLC proliferation, consistent with a larger role of CCR1-binding chemokines in SMLC migration and proliferation and GAD development. CONCLUSIONS: These studies provide a novel mechanistic insight into the formation of vascular intimal hyperplasia and suggest a novel therapeutic strategy for preventing allograft arteriopathy.

Adiponectin inhibits pro-inflammatory signaling in human macrophages independent of interleukin-10.

Macrophages participate pivotally in the pathogenesis of many chronic inflammatory diseases including atherosclerosis. Adiponectin, a vasculoprotective molecule with insulin-sensitizing and anti-atherogenic properties, suppresses pro-inflammatory gene expression in macrophages by mechanisms that remain incompletely understood. This study investigated the effects of adiponectin on major pro-inflammatory signaling pathways in human macrophages. We demonstrate that pretreatment of these cells with adiponectin inhibits phosphorylation of nuclear factor kappaB inhibitor (IkappaB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), induced by either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) alpha, as well as STAT3 phosphorylation induced by interleukin-6 (IL6). Antagonism of IL10 by either neutralizing antibodies or siRNA-mediated silencing did not abrogate the anti-inflammatory actions of adiponectin, indicating that the ability of adiponectin to render human macrophages tolerant to various pro-inflammatory stimuli does not require this cytokine. A systematic search for adiponectin-inducible genes with established anti-inflammatory properties revealed that adiponectin augmented the expression of A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, TNF receptor-associated factor (TRAF) 1, and TNFAIP3-interacting protein (TNIP) 3. These results suggest that adiponectin triggers a multifaceted response in human macrophages by inducing the expression of various anti-inflammatory proteins that act at different levels in concert to suppress macrophage activation.

Novel regulatory effect of angiotensin II type 1 receptor-interacting molecule on vascular smooth muscle cells.

We have recently cloned a novel molecule that interacts with the angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP). In this study, we tested the hypothesis that ATRAP modulates angiotensin II-induced responses in vascular smooth muscle cells. The results of immunoprecipitation and bioluminescence resonance energy transfer assay demonstrated a direct interaction between ATRAP and AT1R at baseline and showed that angiotensin II enhanced the interaction of these proteins >2-fold. The results of immunofluorescence analysis also demonstrated that >65% of ATRAP constitutively colocalized with an endosome marker. Although only 36% of ATRAP colocalized with AT1R at baseline, angiotensin II enhanced the colocalization of these molecules and made 92% of ATRAP colocalize with AT1R on a quantitative fluorescence analysis. Overexpression of ATRAP by adenoviral transfer decreased the cell surface AT1R number from 4.33 to 2.13 fmol/10(6) cells at baseline and from 3.04 to 1.26 fmol/10(6) cells even after removal of angiotensin II. ATRAP also suppressed angiotensin II-mediated increases in c-fos gene transcription and transforming growth factor-beta production. Furthermore, this suppression was accompanied by inhibition of angiotensin II-induced activation of 5-bromodeoxyuridine incorporation. Finally, ATRAP knockdown by small-interference RNA activated angiotensin II-induced c-fos gene expression, which was effectively inhibited by valsartan, an AT1R-specific antagonist. These results indicate that ATRAP promotes internalization of AT1R and attenuates the angiotensin II-mediated c-fos-transforming growth factor-beta pathway and proliferative response in vascular smooth muscle cells, suggesting a novel strategy to inhibit vascular fibrosis and remodeling through a novel and specific blockade of AT1R signaling.

Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo: application of green and red fluorescent protein-expressing retroviral vector.

An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP(+) mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2 x 10(6) GFP(+) EPCs and 2 x 10(5) RFP(+) MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC- and RFP(+) MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.

Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (MSC-Akt) efficiently repaired infarcted rat myocardium and improved cardiac function. Controversy still exists over the mechanisms by which MSC contribute to tissue repair. Herein, we tested if cellular fusion of MSC plays a determinant role in cardiac repair. We injected MSC expressing Cre recombinase, with or without Akt, into Cre reporter mice. In these mice, LacZ is expressed only after Cre-mediated excision of a loxP-flanked stop signal and is indicative of fusion. MSC engraftment within infarcted myocardium was transient but significantly enhanced by Akt. MSC fusion with cardiomyocytes was observed as early as 3 days, but was infrequent, and we found a low rate of differentiation of MSC into cardiomyocytes. MSC-Akt decreased infarct size at 3 days and restored early cardiac function. In conclusion, MSC-Akt improved early repair despite transient engraftment, low levels of cellular fusion, and differentiation. These new observations further confirm our recently reported data that early paracrine mechanisms mediated by MSC are responsible for enhancing the survival of existing myocytes and that Akt could alter the secretion of various cytokines and growth factors.

Angiotensin type 2 receptor is expressed in murine atherosclerotic lesions and modulates lesion evolution.

BACKGROUND: In the vasculature, the angiotensin type 2 (AT2) receptor (AT2R) exerts antiproliferative, antifibrotic, and proapoptotic effects. Normal adult animals have low AT2R expression; however, vascular injury and exposure to proinflammatory cytokines augment AT2R levels. We hypothesized that AT2R expression increases during initiation and progression of atherosclerosis. METHODS AND RESULTS: Atherosclerotic lesions of apolipoprotein (Apo) E(-/-) mice contained AT2Rs, measured by real-time polymerase chain reaction and confirmed by immunohistochemistry. To test the consequences of this expression, male ApoE(-/-), angiotensin II type 2 receptor-deficient (Agtr2-), and ApoE(-/-), wild-type (Agtr2+) mice consumed a high-cholesterol diet from 4 weeks of age. Ten weeks later, overall area and cellular composition of aortic arch lesions did not differ significantly among genotypes. After 16 weeks, ApoE(-/-)/Agtr2+, but not ApoE(-/-)/Agtr2- mice had dramatic decreases in percent positive area of macrophages, smooth muscles, lipids, and collagen. Diminished bromodeoxyuridine incorporation and increased TUNEL staining accompanied these decreases. CONCLUSIONS: Thus, loss of AT2R during the evolution of atherosclerotic lesions augmented the extent of cellularity of atherosclerotic lesions, establishing AT2R as a modulator of atherogenesis.

Searching for transcriptional regulators of Ang II-induced vascular pathology.

Ang II plays a key role in cardiovascular regulation and participates in vascular pathobiology, including inflammation and remodeling. Whether these tissue effects are mediated by direct Ang II actions or indirectly as a result of its influence on hemodynamics is being debated. In vitro data have shown that Ang II induces vascular cellular transcriptional activation and gene expression, but the mechanisms explaining its long-term tissue effects in vivo are relatively unknown. Do the multiple in vivo vascular activities elicited by Ang II (such as inflammation, fibrosis, and vascular cell hypertrophy/proliferation) occur via independent pathways, or do common transcription mechanisms mediate these multiple effects? In this issue, Zhan et al. identify Ets-1 as a critical downstream transcriptional mediator of vascular inflammation and remodeling in vivo; their data suggest that Ets-1 may be a common denominator of a complex process that involves multiple pathways previously considered to be mechanistically independent. Characterization of the critical transcription programs activated by Ang II in vivo and determination of the hierarchy of responses are vital to the understanding of the mechanism of vascular disease and to the development of therapies targeted at inhibiting the common transcription effectors of vascular pathology.

Liver X receptors alpha and beta regulate renin expression in vivo.

The renin-angiotensin-aldosterone system controls blood pressure and salt-volume homeostasis. Renin, which is the first enzymatic step of the cascade, is critically regulated at the transcriptional level. In the present study, we investigated the role of liver X receptor alpha (LXR(alpha)) and LXR(beta) in the regulation of renin. In vitro, both LXRs could bind to a noncanonical responsive element in the renin promoter and regulated renin transcription. While LXR(alpha) functioned as a cAMP-activated factor, LXR(beta) was inversely affected by cAMP. In vivo, LXRs colocalized in juxtaglomerular cells, in which LXR(alpha) was specifically enriched, and interacted with the renin promoter. In mouse models, renin-angiotensin activation was associated with increased binding of LXR(alpha) to the responsive element. Moreover, acute administration of LXR agonists was followed by upregulation of renin transcription. In LXR(alpha) mice, the elevation of renin triggered by adrenergic stimulation was abolished. Untreated LXR(beta) mice exhibited reduced kidney renin mRNA levels compared with controls. LXR(alpha)LXR(beta) mice showed a combined phenotype of lower basal renin and blunted adrenergic response. In conclusion, we show herein that LXR(alpha) and LXR(beta) regulate renin expression in vivo by directly interacting with the renin promoter and that the cAMP/LXR(alpha) signaling pathway is required for the adrenergic control of the renin-angiotensin system.

Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells.

Connector enhancer of KSR (CNK) proteins have been proposed to act as scaffolds in the Ras-MAPK pathway. In this work, using in vivo bioluminescence resonance energy transfer (BRET) assays and in vitro co-immunoprecipitation, we show that hCNK1 interacts with the active form of Rho A (G14V) proteins. The domain of hCNK1 that allows binding to Rho proteins involves the C-terminal PH domain. Overexpression of hCNK1 does not affect the actin cytoskeleton and does not modify the appearance of stress fibers in cells overexpressing a constitutively active form of RhoA. In contrast, hCNK1 was able to significantly decrease the RhoA-induced transcriptional activity of the serum response element (SRE) without effect on the Ras-induced SRE activation. These results identify hCNK1 as a specific partner of Rho proteins both in vitro and in vivo and suggest a role of hCNK1 in the signal transduction of Rho proteins.

Identification of calcium-modulating cyclophilin ligand (CAML) as transducer of angiotensin II-mediated nuclear factor of activated T cells (NFAT) activation.

Angiotensin II (Ang II) plays a central role in cardiovascular physiology and disease. Ang II type I receptor (AT1) is thought to mediate most actions of Ang II. A novel AT1 receptor intracellular partner called AT1 receptor-associated protein (ATRAP) was identified, but its exact function has not been elucidated. A yeast two-hybrid screen using ATRAP as bait identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner. Yeast two-hybrid and coimmunoprecipitation analysis demonstrated that the N-terminal hydrophilic domain of CAML (amino acids (aa) 1-189) mediates a specific interaction between ATRAP and CAML. Our analysis also showed that aa 40-82 of ATRAP contribute to this interaction. Bioluminescence resonance energy transfer and intracellular colocalization analysis by immunofluorescence in HEK293 cells verified this association within endoplasmic reticulum vesicular structures. Functionally, transcriptional reporter assays and RNA interference ATRAP experiments demonstrated that ATRAP knockdown increased nuclear factor of activated T cells (NFAT) activity. Overexpression of ATRAP decreased Ang II- or CAML-induced NFAT transcriptional activation, whereas an ATRAP-interacting domain of CAML (aa 1-189) sensitized NFAT activation in response to Ang II. These results indicate that CAML is an important signal transducer for the actions of Ang II in regulating the calcineurin-NFAT pathway and suggest that the interaction of CAML with ATRAP may mediate the Ang II actions in vascular physiology.

Discs large (Dlg1) complexes in lymphocyte activation.

T cell antigen recognition involves the formation of a structured interface between antigen-presenting and T cells that facilitates the specific transmission of activating and desensitizing stimuli. The molecular machinery that organizes the signaling molecules and controls their disposition in response to activation remains poorly understood. We show here that in T cells Discs large (Dlg1), a PDZ domain-containing protein, is recruited upon activation to cortical actin and forms complexes with early participants in T cell activation. Transient overexpression of Dlg1 attenuates basal and Vav1-induced NFAT reporter activation. Reduction of Dlg1 expression by RNA interference enhances both CD3- and superantigen-mediated NFAT activation. Attenuation of antigen receptor signaling appears to be a complex, highly orchestrated event that involves the mutual segregation of important elements of the early signaling complex.