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Purpose: Age-related macular degeneration (AMD) is a chronic and progressive macular degenerative disease that culminates in a gradual deterioration of central vision. Despite its prevalence, the key biomarkers for AMD have not yet been fully elucidated. In this study, we aimed to efficiently identify biomarkers crucial for diagnosing AMD. Methods: Three datasets pertaining to retinal pigment epithelium (RPE)/choroid tissues associated with AMD were selected from the GEO database. The GSE50195 dataset was utilized to conduct weighted gene co-expression network analysis (WGCNA) for identifying module genes linked to AMD. KEGG and GO enrichment analyses were subsequently conducted on these module genes. GSE29801 and GSE135092 datasets were subjected to differential expression analysis to pinpoint the DEGs intersecting with the module genes. Subsequently, wet AMD (wAMD) and dry AMD (dAMD) mouse models were developed, from which RPE/choroid tissues were harvested to validate the hub genes via RT-qPCR and Western blot. Results: Using the WGCNA, we selected the "antiquewhite4" module (r = 0.91 and p = 7e-07), which contains a total of 325 genes. Through the intersection of module genes with DEGs, nine hub genes were identified. Pathways involved in complement and coagulation cascades, ECM-receptor interactions, unsaturated fatty acid biosynthesis, and fatty acid elongation play important roles in AMD. Notably, CDH18 demonstrated notable variance across all three datasets. Post validation using RT-qPCR experiments revealed a significant downregulation of CDH18 in both dAMD and wAMD. EGLN3 was expressed at low levels in wAMD. In dAMD, EYA2, LTB, and PODXL were significantly downregulated, whereas APOC1 was notably upregulated. Western blot confirmed that CDH18 was lowly expressed in dAMD and wAMD mouse models. Conclusion: CDH18 was identified as the key gene involved in the pathogenesis of AMD. An imbalance of the complement and coagulation cascades is a potential mechanism of AMD. This study provides a novel idea for diagnosing and treating AMD in the future.

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Postoperative Atrial Fibrillation (POAF) frequently follows Coronary Artery Bypass Grafting (CABG) surgery. This prospective study investigates genes linked to POAF in CABG patients, aiming to create a predictive model. Employing differential gene and methylation analyses, the study identified four genes (WARS2, CKAP2, CHI3L1, HSD17B6) associated with POAF. Preoperative plasma samples and clinical data were collected from 139 CABG patients, categorized into POAF (+) (43) and POAF (-) (96). Real-time quantitative PCR assessed gene expression, and a predictive model using the LASSO method demonstrated robust performance, with AUC values of 0.8895 in the training set and 0.7840 in the test set. This pioneering study integrates genomics and clinical data, suggesting WARS2, CKAP2, and CHI3L1 as potential indicators for POAF prediction.

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BACKGROUND: Ginsenoside Rh2 (Rh2) is a major biological component of ginseng that exerts antitumor activities in multiple cancers including Non-Small Cell Lung Cancers (NSCLCs). Rh2 also enhances the anti-tumor effects of various chemotherapy drugs including cisplatin at relatively low concentrations. Here, the mechanistic role of Rh2 in chemotherapy-treated NSCLCs will be investigated. METHODS: In this study, FACS, western blot and siRNA addition were used to analyze the role of Rh2 in cisplatin- treated lung adenocarcinoma A549 and H1299 cells. RESULTS: Subsequent observations indicated that Rh2 enhanced cisplatin-induced NSCLCs A549 and H1299 cells apoptosis. Cisplatin-induced productive autophagy was repressed by Rh2 in A549 cells. Rh2 also enhanced cisplatin cytotoxicity by elevating superoxide dismutase activity and repressing cisplatin-induced superoxide generation. Conversely, Rh2 was found to repress cisplatin-induced phosphorylation of epidermal growth factor receptor, phosphoinositide 3-kinase, protein kinase B, and autophagy. Cisplatin-induced Programmed Death- Ligand 1 (PD-L1) expression was repressed by Rh2 via the superoxide. CONCLUSION: These findings suggest that Rh2 enhanced the function of cisplatin by repressing superoxide generation, PD-L1 expression, and autophagy in lung adenocarcinoma cells.

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BACKGROUND: Ginsenoside Rh2 (Rh2), which is extracted from ginseng, exerts antitumor activity. Here we would like to study the role of Rh2 on hypoxia-induced migration in lung adenocarcinoma. METHODS: Lung adenocarcinoma A549 and H1299 cells were cultured in 1% O2 condition to mimic the hypoxic tumor microenvironment. The migrations of cancer cells were measured by transwell assay and scratch assay. RESULTS: Rh2 could inhibit hypoxia-induced A549 and H1299 cell migration via increase of mir-491 expression. Further, mir-491 antisense oligonucleotide could repress hypoxia-induced migration and the expression of matrix metalloproteinase (MMP)-9 expression in Rh2-treated A549 cells. CONCLUSION: These findings suggest that Rh2 exerts anti-metastasis activity in the hypoxic tumor microenvironment in lung adenocarcinoma cells via mir-491.

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Therapeutic strategies based on stem cells have been shown to have potential in improving the condition of severe lung diseases. In this study, the suppressive effects of conditioned medium (CM) of induced pluripotent stem cells (iPSCs) on pulmonary fibroblast differentiation were investigated in a series of in vitro and in vivo experiments. Moreover, the underlying mechanisms through which iPSC-CM inhibited the differentiation of fibroblasts into myofibroblasts were explored as well. iPSCs were generated using a mouse 3-gene transfection method, myofibroblast-like cells were induced by incubating human fibroblasts with transforming growth factor-ß1 (TGF-ß1) and mouse models of pulmonary fibrosis (PF) were established by an injection of bleomycin. Based on these experiments, the effects of iPSC-CM on collagen accumulation, lung structure and the TGF-ß1-mediated pathway were determined. It was found that treatment with iPSC-CM markedly reduced the proliferation of TGF-ß1-exposed cells, and the activities of TGF-ß1, Smad-2 and Smad-3. Accompanied by alterations in the expression of the indicated molecules, the lung structure of mice with PF was also markedly ameliorated. The present study confirmed the protective effects of iPSC-CM on lung tissue against PF, and it was also inferred that the ameliorating function of iPSC-CM on PF may be exerted through the blocking of TGF-ß1/Smad signal transduction pathway.

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Pulmonary fibrosis is a progressive and irreversible fibrotic lung disorder with high mortality and few treatment options. Recently, induced pluripotent stem (iPS) cells have been considered as an ideal resource for stem cell-based therapy. Although, an earlier study demonstrated the therapeutic effect of iPS cells on pulmonary fibrosis, the exact mechanisms remain obscure. The present study investigated the effects of iPS cells on inflammatory responses, transforming growth factor (TGF)-ß1 signaling pathway, and epithelial to mesenchymal transition (EMT) during bleomycin (BLM)-induced lung fibrosis. A single intratracheal instillation of BLM (5 mg/kg) was performed to induce pulmonary fibrosis in C57BL/6 mice. Then, iPS cells (c-Myc-free) were administrated intravenously at 24 h following BLM instillation. Three weeks after BLM administration, pulmonary fibrosis was evaluated. As expected, treatment with iPS cells significantly limited the pathological changes, edema, and collagen deposition in lung tissues of BLM-induced mice. Mechanically, treatment with iPS cells obviously repressed the expression ratios of matrix metalloproteinase-2 (MMP-2) to its tissue inhibitor -2 (TIMP-2) and MMP-9/TIMP-1 in BLM-induced pulmonary tissues. In addition, iPS cell administration remarkably suppressed BLM-induced up-regulation of pulmonary inflammatory mediators, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and prostaglandin E2. We further demonstrated that transplantation of iPS cells markedly inhibited BLM-mediated activation of TGF-ß1/Mothers against decapentaplegic homolog 2/3 (Smad2/3) and EMT in lung tissues through up-regulating epithelial marker E-cadherin and down-regulating mesenchymal markers including fibronectin, vimentin and α-smooth muscle actin. Moreover, in vitro, iPS cell-conditioned medium (iPSC-CM) profoundly inhibited TGF-ß1-induced EMT signaling pathway in mouse alveolar epithelial type II cells (AECII). Collectively, our results suggest that transplantation of iPS cells could suppress inflammatory responses, TGF-ß1/Smad2/3 pathway and EMT during the progression of BLM-induced pulmonary fibrosis, providing new useful clues regarding the mechanisms of iPS cells in the treatment for this disease.

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OBJECTIVE: To investigate the value of lymphocyte count in assessing cellular immune function in patients with community-acquired pneumonia. METHODS: Ninety-three patients with community-acquired pneumonia (including 53 non-severe and 40 severe cases) and 52 healthy adults were examined for routine blood test and T lymphocyte count. Blood lymphocyte counts and absolute T lymphocyte counts were compared among the 3 groups and their correlation was analyzed. RESULTS: Compared with the healthy control subjects, patients with community-acquired pneumonia showed significantly lower blood lymphocyte counts and CD3(+), CD4(+), and CD8(+) levels (P<0.05). CD3(+), CD4(+), and CD8(+) levels were positively correlated with blood lymphocyte counts. With blood lymphocyte count as the independent variable (L), and the regression equations for CD3(+), CD4(+), and CD8(+) levels were CD3(+)=485.45L+313.48 (F=59.68, P<0.01), CD4(+)=192.57L+290.11 (F=24.62, P<0.01), and CD8(+)=275.14L+18.04 (F=23.46, P<0.01) in the control group; CD3(+)=564.15L+25.04 (F=96.56, P<0.01), CD4(+)=381.91L-37.45 (F=68.60, P<0.01), and CD8(+)=165.61L+61.83 (F=55.47, P<0.01) in non-severe pneumonia group; and CD3(+)=565.44L+49.09 (F=31.87, P<0.01), CD4(+)=332.34L-17.37 (F=43.64, P<0.01), and CD8(+)=223.46L+54.39 (F=13.90, P<0.01) in severe pneumonia group. CONCLUSION: Patients with community-acquired pneumonia have decreased cellular immune function. Absolute T lymphocyte count can be estimated by blood lymphocyte count to save the cost of laboratory tests.

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The present research assessed the effects of lettuce glycoside B (LGB), a compound separated and purified from Pterocypsela laciniata, on irradiation-induced pulmonary fibrosis and explored the mechanism involved. Animal model of irradiation exposure inducing pulmonary fibrosis was established by Co irradiator. Rats were intraperitoneally treated with LGB (100, 200 and 400 mg/kg) once per day for a month. Lung index data were analyzed. The levels of fibrosis were assessed by hydroxyproline (Hyp) of pulmonary and lung tissue sections after irradiation exposure. Alveolitis and fibrosis levels were calculated from semi-quantitative analysis of hematoxylin and eosin and Masson's trichrome lung section staining. The serum levels of transforming growth factor ß1 (TGF-ß1), interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) were also evaluated. Antioxidant enzymes of superoxide dismutase (SOD) were measured in serum. Moreover, we also measured serum malondialdehyde (MDA) levels, a marker of oxidative stress. Treatment with LGB significantly reduced mortality rates and lung index scores and MDA content, enhanced SOD and other antioxidant enzymes activity, and regulated serum levels of TGF-ß1, IL-6, and TNF-α. These results demonstrated that LGB significantly inhibited irradiation-induced pulmonary fibrosis. Furthermore, the results suggested promising clinical effect of LGB therapies for treating irradiation-induced pulmonary fibrosis.

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To investigate the influence of NF-κB antisense oligonucleotide on transdifferentiation of fibroblast in the pathological process of bleomycin-induced pulmonary fibrosis in mice. 6 h before molding of C57BL/6 model of pulmonary fibrosis in mice, NF-κB antisense oligonucleotide was injected from caudal vein. Then the lung tissue was collected for primary culture as well as model group and control group. Cultured cells were used for immunocytochemical staining of p65, IκB-α and α-SMA proteins as well as in situ hybridization staining of p65 and IκB-α. Then image analysis was carried out. The expressions of all the indicators were expressed as mean optical density. Compared with the control group, the expressions of p65 protein, IκB-α protein and α-SMA protein of model group were increased, as well as the expressions of p65 mRNA and IκB-α mRNA (P < 0.05). Compared with model group, the expressions of all indicators of intervention group were decreased (P < 0.05). P65 protein and p65 mRNA were positively correlated with the expression of α-SMA protein respectively. p65 protein and p65 mRNA were positively correlated with the expressions of IκB-α protein and IκB-α mRNA respectively. NF-κB antisense oligonucleotide can inhibit the transdifferentiation of fibroblast towards myofibroblast in the pathological process of bleomycin-induced pulmonary fibrosis in mice.

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OBJECTIVE: NF-κB, especially p65 subunit, plays important role in the process of pulmonary fibrosis. In this study, we transformed fibroblast into myofibroblast induced by bleomycin, and then studied the effects of NF-κB p65 antisense oligonucleotide on pulmonary fibrosis in mouse model. METHODS: Pulmonary fibrosis was induced by bleomycin in C57BL/6 mouse (modeling group). The NF-κB antisense oligonucleotide was injected intravenously into mouse 6 hours before inducing (test group), we performed broncho-alveolar lavage and blood collecting through cardiac puncture. Bronchoalveolar Lavage Fluid (BALF) and serum from normal C57BL/6 mouse (control group) were collected for comparison. Immunohistochemistry staining of the NF-κB and α-SMA on lung tissues and cultured cells were carried out in each group, respectively. RESULTS: The expression level of NF-κB and α-SMA were both consistently higher in modeling group when compared with control group (P < 0.05). Meanwhile, they were reduced significantly through the intervention of NF-κB p65 antisense oligonucleotide in the test group (P < 0.05). More importantly, the expression of NF-κB was positively correlated with α-SMA. CONCLUSION: our study suggests the potential in prevention of bleomycin-induced pulmonary fibrosis with NF-κB p65 antisense oligonucleotide.

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Influenza virus can cause an acute respiratory illness of variable intensity. In our study, we describe the clinical and immunological characteristics of pregnant and nonpregnant women who were hospitalized with 2009 H1N1 influenza and pneumonia in Shenyang, China, from November 2009 to January 2010. Forty-two female patient infected with H1N1 were divided into groups according to pregnancy. Clinical data were collected. Cytokines and anti-H1N1 IgG subclasses were detected. We observed significant lymphopenia, hypoproteinemia, reduced CD4(+)T cell counts and CD4(+)/CD8(+) ratios, reduced anti-H1N1 IgG subclasses IgG2 and IgG3 constituent ratios, elevated C reactive protein and interleukin-10 levels with regard to nonpregnant H1N1 group. Compared with the healthy pregnant group, the pregnant H1N1 group showed elevated aspartate aminotransferase and glutamic alanine aminotransferase levels, an increased interferon-gamma and interleukin-10 levels and reduced anti-H1N1 IgG subclasses IgG2, IgG3 and IgG4 combination ratios. There was a statistically significant association between imbalanced anti-H1N1 immunoglobulin subclasses and dysregulated cytokines in pregnant women with H1N1 infection.

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The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFß signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFß expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFß expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFß-mediated fibrogenesis. We conclude that TGFß-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

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Lysophosphatidic acid (LPA) is a potent bioactive lysophospholipid. Accumulated evidence supports a role for LPA in inflammation. To profile LPA-induced cytokine production in vascular smooth muscle cells (SMCs), we used a cytokine antibody array system and found that LPA prominently induces the secretion of IL-6 and monocyte chemoattractant protein (MCP)-1 from human aortic SMCs (HASMCs). The mechanism by which LPA induces MCP-1 expression in SMCs has been previously reported. However, LPA induction of IL-6 secretion from vascular SMCs and its regulatory mechanism are unknown. The present study reveals that LPA induces the expression of IL-6 mRNA and protein in HASMCs as well as the secretion of IL-6 protein in a time-dependent manner. Our results demonstrate that LPA-specific receptor 1 (LPA(1)) mediates LPA-induced IL-6 secretion and that LPA induction of IL-6 is independent of the EGF receptor pathway. Our data further show that PKC-mediated p38 MAPK is responsible for the IL-6 secretion. Finally, small interfering RNA depletion experiments revealed that p38alpha is specifically responsible for the LPA-induced IL-6 secretion. The present study profiles the regulatory relationship between LPA and multiple cytokines in vascular SMCs for the first time, provides the first evidence that LPA upregulates IL-6 in vascular SMCs, and reveals the regulatory mechanism of LPA-induced IL-6 production in HASMCs. In light of the emerging roles of LPA and IL-6 in vascular inflammation, the understanding of the regulatory mechanism may contribute to the treatment and prevention of cardiovascular disorders.

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OBJECTIVE: Monocyte activation and migration are crucial events in the development of atherosclerosis and other inflammatory diseases. This study examined the role of protein kinase D (PKD) in monocyte migration. Method and Results- PKD2 is the predominant isoform of PKD expressed in monocytic THP-1 cells and primary human monocytes. Lysophosphatidylcholine (lysoPC), a prominent component of oxidized low-density lipoprotein, induces rapid and marked PKD activation in these cells. Using multiple approaches, including dominant-negative mutants and small interfering RNA knock-down, we found that lysoPC-induced PKD2 activation was required for the activation of both ERK and p38 MAPK. p38 MAPK mediation of lysoPC-induced monocytic cell migration was reported previously; our results reveal that the lysoPC-induced PKD2-p38 pathway controls monocyte migration. CONCLUSIONS: This study provides the first evidence that (1) lysoPC activates PKD, (2) PKD2 has a novel role in p38 activation, and (3) the PKD2-activated p38 pathway is responsible for lysoPC-induced migration of THP-1 cells and human monocytes. Thus, PKD is a novel and functional intracellular regulator in both lysoPC signaling and monocyte migration. These results suggest a new role for PKD2 in the development of atherosclerosis and other inflammatory diseases.

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Histamine, a potent inflammatory mediator, has multiple effects on the pathogenesis of atherosclerosis. This study investigates the effect of histamine on the expression of early growth response factor 1 (Egr-1), a master transcription factor that regulates the expression of an array of atherogenic genes in atherosclerotic lesions. Histamine markedly and rapidly induces Egr-1 mRNA and protein expression in primary human aortic endothelial cells (HAECs). Histamine-induced Egr-1 expression is dependent on the activation of the H1 receptor. Histamine also rapidly and transiently activates protein kinase C-delta (PKCdelta), extracellular signal-regulated kinase (ERK)1/2, p38 kinase, and c-Jun N-terminal kinase (JNK) prior to Egr-1 induction. Using specific pharmacological inhibitors and small interfering RNA technology, we determined that PKCdelta and ERK, but not p38 and JNK, mediate histamine-induced Egr-1 expression. Our data provide the first evidence that histamine regulates expression of Egr-1 in mammalian cells and demonstrate a novel role of PKCdelta in up-regulation of Egr-1 expression. The present study reveals the following regulatory mechanism: histamine up-regulates Egr-1 expression in primary HAECs via the H1 receptor and the PKCdelta-dependent ERK activation pathway. Our data also imply that CREB, a downstream component of the ERK pathway, regulates Egr-1 expression in HAECs. Importantly, these results suggest a central role of Egr-1 in histamine-induced gene expression and in histamine-induced vascular disease.

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Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA(1) receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of alpha, beta, gamma and delta, we have identified that the activation of ERK2 (p42) and p38alpha, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38alpha in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA(1) mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA(1), ERK2 and p38alpha are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.

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OBJECTIVE: Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein, is a potent bioactive phospholipid. Early growth response gene-1 (Egr-1), an important transcription factor, regulates expression of an array of genes involved in vascular diseases. Whether and how LPA regulates the transcriptional machinery of Egr-1 gene is unknown and is addressed in this study. METHOD AND RESULTS: We found that LPA markedly induces Egr-1 mRNA and protein in aortic smooth muscle cells (SMCs). RNA stability and nuclear run-on assays reveal that LPA-induced Egr-1 gene expression is controlled at the transcriptional level. Reporter gene analyses have shown that the -141 to +20 nt region of the Egr-1 promoter contains regulatory elements. Electrophoretic mobility shift assays reveal that the DNA-binding activities of both CREB and SRF to the CRE and SRE motifs of the Egr-1 promoter are markedly elevated in response to LPA. The increased binding activity depends on the phosphorylation of CREB and SRF. Luciferase assays of a series of deleted or mutated Egr-1 promoter-reporter gene constructs, along with dominant negative CREB transfection analysis revealed that the 2 CRE sites and the 2 proximal SRE sites in the Egr-1 promoter are required for maximal LPA-induced Egr-1 gene expression. CONCLUSIONS: Our data reveal that LPA regulates Egr-1 expression via transcription factors CREB and SRF. These results establish a novel role for CREB in mediating LPA-induced gene expression. Our results imply that elevated LPA levels may, through activation of Egr-1, which regulates an array of atherogenic genes, exacerbate atheromatous lesions.

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OBJECTIVE: Angiotensin II (Ang II), through its specific signaling cascades, exerts multiple effects on vascular smooth muscle cells (SMCs). It has been shown that Ang II stimulates activation of protein kinase D (PKD), a member of a new class of serine-threonine kinases. However, little is known regarding the upstream cascade of the intracellular signaling that leads to PKD activation. In the present study, we investigated upstream molecules that mediate Ang II-induced PKD activation in SMCs. METHODS AND RESULTS: Protein kinase C (PKC) inhibitors completely block Ang II-induced PKD activation, and pretreatment with phorbol 12,13-dibutyrate downregulates Ang II-induced PKD activation, indicating that classical or novel isoforms of PKC mediate Ang II-induced PKD activation. Furthermore, the finding that rottlerin, a PKCdelta-specific inhibitor, blocks PKD activation suggests that PKCdelta, a member of novel PKCs, mediates Ang II-induced PKD activation. By using dominant-negative approaches, our results demonstrate that expression of the dominant-negative PKCdelta, but neither the dominant-negative form of PKCepsilon nor PKCzeta, inhibits PKD activation. These results further substantiate the finding that Ang II-induced PKD activation is mediated by PKCdelta. Moreover, using selective Ang II receptor antagonists, our data show that the Ang II type 1 (AT1) receptor but not the AT2 mediates Ang II-stimulated PKD activation. CONCLUSIONS: This study reveals for the first time that Ang II-induced PKD activation is mediated via AT1 and regulated by PKCdelta in living cells. These data may provide new insights into molecular mechanisms involved in Ang II-induced physiological and pathological events.

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Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.

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