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Objective: Although a new matrix formulation fentanyl has been used throughout the world for cancer pain management, few data about its efficacy and clinical outcomes associated with its use in Chinese patients have been obtained. This study aimed to assess the efficacy and safety of the new system in Chinese patients with moderate to severe cancer pain., Methods: A total of 474 patients with moderate to severe cancer pain were enrolled in this study and were treated with the new transdermal fentanyl matrix patch (TDF) up to 2 weeks. All the patients were asked to record pain intensity, side effects, quality of life (QOL), adherence and global satisfaction. The initial dose of fentanyl was 25 μg/h titrated with opioid or according to National Comprehensive Cancer Network (NCCN) guidelines. Transdermal fentanyl was changed every three days., Results: After 2 weeks. The mean pain intensity of the 459 evaluated patients decreased significantly from 5.63±1.26 to 2.03±1.46 (P<0.0001). The total remission rate was 91.29%, of which moderate remission rate 53.16%, obvious remission rate 25.49% and complete remission rate 12.64%. The rate of adverse events was 33.75%, 18.78% of which were moderate and 3.80% were severe. The most frequent adverse events were constipation and nausea. No fatal events were observed. The quality of life was remarkably improved after the treatment (P<0.0001)., Conclusion: The new TDF is effective and safe in treating patients with moderate to severe cancer pain, and can significantly improve the quality of life.

Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17-producing cells, has recently been shown to be a proinflammatory cytokine involved in chronic inflammation and autoimmune disease. In this study, we report that IL-17A increased the synthesis and secretion of collagen and promoted the epithelial-mesenchymal transition in alveolar epithelial cells in a TGF-β1-dependent manner. Using in vivo fibrotic models, we found IL-17A expression to be elevated and IL-17A-associated signaling pathways to be activated in fibrotic lung tissues. Neutralization of IL-17A in vivo promoted the resolution of bleomycin-induced acute inflammation, attenuated pulmonary fibrosis, and increased survival. Additionally, IL-17A antagonism inhibited silica-induced chronic inflammation and pulmonary fibrosis. Targeting IL-17A resulted in a shift of the suppressive immune response in fibrotic lung tissue toward a Th1-type immune response, and it effectively induced autophagy, which promoted the autophagic degradation of collagen and autophagy-associated cell death. Moreover, IL-17A was found to attenuate the starvation-induced autophagy, and autophagy modulators regulated collagen degradation in the alveolar epithelial cells in a TGF-β1-independent manner. Administration of 3-methylamphetamine, an autophagy inhibitor, reversed the therapeutic efficacy of IL-17A antagonism in pulmonary fibrosis. Our studies indicate that IL-17A participates in the development and progression of pulmonary fibrosis in both TGF-β1-dependent and -independent manners and that the components of the IL-17A signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases.

Aim: To explore the pathogenic role of Th17 cells and interleukin-17A (IL-17A)-associated signaling pathways in spontaneous pulmonary emphysema induced by a Toll-like receptor 4 mutant (TLR4(mut))., Methods: Lungs were obtained from wild-type (WT) or TLR4mut mice that were treated with or without recombinant mouse IL-17A (1 μg·kg(-1)·d(-1), ip) from the age of 3 weeks to 3 months. Pulmonary emphysema was determined using histology, immunochemistry, and biochemical analysis. T cell polarization was determined with flow cytometry, the levels of cytokines were measured using ELISA, and the levels of IL-17A-associated signaling molecules were detected using Western blot., Results: Compared to WT mice, 3 month-old TLR4(mut) mice were characterized by significantly reduced infiltration of Th17 cells into lungs (2.49%±1.13 % νs 5.26%±1.39%), and significantly reduced expression levels of IL-17A (3.66±0.99 pg/μg νs 10.67±1.65 pg/μg), IL-23 (12.43±1.28 pg/μg νs 28.71±2.57 pg/μg) and IL-6 (51.82±5.45 pg/μg νs 92.73±10.91 pg/μg) in bronchoalveolar lavage fluid. In addition, p38 MAPK phosphorylation and AP-1 expression were decreased to 27%±9% and 51%±8%, respectively, of that in WT mice. Treatment of TLR4(mut) mice with IL-17A increased the infiltration of Th17 cells into lungs and expression levels of IL-17A, IL-6, and IL-23 in bronchoalveolar lavage fluid, attenuated MDA and apoptosis, and improved emphysema accompanied with increased phosphorylation of p38 MAPK and expression of AP-1., Conclusion: Th17 cells, in particular the cytokine IL-17A, play a crucial role in the pathogenesis of TLR4(mut)-induced spontaneous pulmonary emphysema. Both of them are potential targets for therapeutic strategies for pulmonary emphysema.

Background: Diagnosis and treatment for respiratory symptoms (RSs) of gastroesophageal reflux disease (GERD) is more difficult than that for common esophageal symptoms. The goal of this study was to evaluate the efficacy and safety of radiofrequency (RF) treatment on RSs of GERD in a preliminary 12-month follow-up observation., Methods: From April 2006 to October 2008, 505 GERD patients with mainly respiratory presentations such as wheezing, chronic cough or hoarseness, were treated by endoscopic RF. A questionnaire was completed before and after treatment, using a six-point scale ranging from 0 to 5 to assess symptom severity and frequency. The symptom score was the sum of frequency and severity., Results: Symptom scores were significantly improved at the end of the follow-up period. The mean heartburn score decreased from 5.31 to 1.79. The mean regurgitation score decreased from 5.02 to 1.64; mean cough score decreased from 6.77 to 2.85; mean wheezing score decreased from 7.83 to 3.07; and mean hoarseness score decreased from 5.13 to 1.81 (P < 0.01). No major complications or deaths occurred. Minor complications included temporary post-procedural retrosternal unease or pain (n = 106; 21.0%), mild fever (n = 86; 17.0%), transient nausea/vomiting (n = 97; 19.2%), and transient dysphagia (n = 42; 9.3%). Thirty-five (6.9%) patients had recurrence of symptoms. Endoscopic RF treatment was repeated in six patients, and laparoscopic fundoplication was performed in seven., Conclusion: Endoscopic RF is an effective and safe means to treat RSs in patients with GERD.

Background: Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown., Methods and Findings: Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis., Conclusion and Significance: Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.

Aim: to study whether activation of TLR9 by CpG-ODN would protect against and/or reverse renal fibrosis., Methods: animals were treated with CpG-ODN before or after undergoing a unilateral ureteral obstruction (UUO) procedure. The interstitial fibrotic lesions of obstructed kidneys were evaluated using histology and immunohistostaining. The Th2-type cytokine profile and the expression and activity of sma and mad related protein (Smad)3, signal transducers and activators of transcription (Stat)3, extracellular regulated protein kinases (ERK), and p38 kinase were determined using RT-PCR or Western blot., Results: the obstructed kidneys displayed a significant increase in interstitial fibrosis, an infiltration of macrophages in the interstitium, and an enhanced expression of Th2 cytokines. Prophylactic application of CpG-ODN (40 microg/kg every 3 days from 2 h before UUO until the 14th day after UUO) suppressed the expression of α-smooth muscle actin, collagen deposition, and hydroxyproline in the UUO kidneys of rats. Moreover, CpG-ODN not only decreased the infiltration of macrophages but also inhibited the expression of chemokines CCL2 and CCL5, the Th2 cytokine IL-13, and the profibrogenic cytokines transforming growth factor (TGF)-β1 and plasminogen activator inhibitor (PAI)-1 in UUO kidneys of rats. Importantly, therapeutic administration of CpG-ODN (10 microg/mouse, ip, every 3 days from the 4th day to 21st day after UUO) reversed the established renal fibrosis, which was accompanied by significant reductions in the activity of ERK, Smad3, and Stat3 and an increase in the activity of p38 kinase., Conclusion: the activation of TLR9 by CpG-ODN attenuates UUO-induced renal fibrosis by reversing an immunosuppressive microenvironment in the fibrotic renal tissue, which might be a novel therapeutic strategy against fibrotic renal diseases.

Aims: Innate and adaptive immune responses are associated with the development of hypertension-induced myocardial hypertrophy and fibrosis. As a result, we investigated whether heat shock protein (HSP) 70, which is a molecule of damage-associated molecular patterns, could induce inflammation in the myocardium and promote the development of hypertension-induced cardiac hypertrophy and fibrosis., Methods and Results: We found that HSP70 serum levels, as well as the amount of HSP70 translocation to the cardiomyocyte membranes and the interstitial space, were elevated in the hypertensive mice caused by abdominal aortic constriction (AAC). Transcriptional inhibition of HSP70 expression by a specific heat shock transcript factor inhibitor, KNK437, reduced the serum level, and the re-distribution of HSP70. It promoted myocardial hypertrophy and cardiac dysfunctions although it protected animals from AAC-induced cardiac fibrosis. On the other hand, the functional antagonism of HSP70 by an anti-HSP70 antibody attenuated AAC-induced cardiac hypertrophy and fibrosis without adverse haemodynamic effects. The cardioprotective effect of the anti-HSP70 antibody was largely attributed to its ability to block AAC-activated immune response in the heart, as was indicated by suppressing the hypertension-enhanced conjugation of HSP70 with toll-like receptor 4, reducing heart-infiltrating macrophages, decreasing the expression of pro-inflammatory factor monocyte chemoattractant protein-1 and profibrotic factor transforming growth factor beta 1, and attenuating pro-hypertrophy signal MAPK P38 and ERK., Conclusion: These results indicate that intracellular and extracellular HSP70 have different roles in the regulation of cardiac remodelling and function in response to hypertension. Extracellular HSP70 is a potential therapeutic target against cardiac hypertrophy and fibrosis.

Abnormal cardiovascular tissue remodeling characterized by myocardial hypertrophy, myocardial cell loss and myocardial fibrosis, are the primary pathological changes for multiple cardiovascular diseases. Targeting tissue remodeling using small molecules (e.g., relaxin, KNK437), biological agents (e.g., BCG, anti-TLR2 antibody), or herb medicine complex CFX and so on, can attenuate tissue fibrosis and improve cardiac functions. Therefore, regulating the property of inflammation and reversing tissue fibrosis should promote the formation of optimal tissue environments for stem cells mobilization and proliferation, which enhances the myocardial regeneration and is potential therapeutic strategy against chronic cardiovascular diseases.

Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.

The method of determination of iron, titanium and vanadium in indissolvable vanadium and titanium magnetite has been established by inductively coupled plasma atomic emission spectroscopy through adding the complexant A and using microwave-assisted digestion. The optimal conditions are confirmed by orthogonal experiment: 0.1 g vanadium and titanium magnetite, 0.04 g complexant A, 12 mL concentrated HC1, 10 min digestion time, and 385 W microwave power. The newly-established method has been applied to digest vanadium and titanium magnetite of Panzhihua Iron and Steel Institute (GBW07226). The iron, titanium and vanadium were detected by ICP-OES, and both comparative error (Er%) and comparative standard deviation (RSD%) met the demand of analytical chemistry, and the complexant A can significantly accelerate the dissolution of vanadium and titanium magnetite through the complexation with the dissolved metal ions, and making the surface of sample and hydrochloric acid medium to update constantly. The determination of the main and trace elements of digestion solution at the same time was achieved by ICP-OES. The method has the advantages of less use of reagents, economy, rapidness, and being friendly to environment, and it meets the requirement for rapid and volume determination. So the method has the value of practical application for the entry-exit inspection and quarantine department of the state and other relevant inspection units.

Recent studies indicate that increasing protein synthesis in cells beyond the capacity for folding of nascent polypeptides or accumulation of unfolded/misfolded proteins within the endoplasmic reticulum (ER) lumen will disrupt ER homeostasis and trigger the unfolded protein response (UPR). ER stress or UPR is not only critical for cell homeostasis and embryogenesis, but ERS/UPR can initiate inflammatory responses in specialized cells and tissues to participate in the pathogenesis of inflammatory diseases. Here, we summarize recent progresses in the roles of UPR and inflammation coupling in the pathogenesis of human chronic diseases, by which may result in approaches to manipulate ERS-UPR-inflammation and to provide therapeutic opportunity for the prevention and treatment of chronic diseases.

Transforming growth factor-beta1 (TGF-beta1) regulates a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Here we report that death effector domain-containing DNA-binding protein (DEDD) physically interacts with Smad3. The inhibition of Smad3 by DEDD resulted in a reduction in TGF-beta1/Smad3-mediated transcription. DEDD inhibited the functions of Smad3 by preventing Smad3 phosphorylation, which led to the reduced expression of TGF-beta1/Smad3-targeted genes. TGF-beta1 inhibited DEDD expression, and DEDD inhibited TGF-beta1-mediated invasion. Therefore, our findings suggest that through its interaction with Smad3, DEDD is a novel negative regulator of the TGF-beta1 signaling pathway., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The interaction of tumor cells with host cells undergoes a progress of immunoediting, including surveillance, equilibrium and escape. The capability of escape from immune-surveillance is a hall marker of tumor cells, which greatly contributes to the tumor growth out of control and the therapy failure in tumor metastasis. It is indicated that tumor cells can recruit amounts of immune cells to tumor site and establish a suppressive immune microenvironment leading to tumor escape. The critical factor for tumor immunotolerance is the establishment of immunosuppressive microenvironment by interaction between pattern recognition receptors with pathogen-associated molecular patterns and damage-associated molecular patterns released from tumor tissue. Therefore, targeting tumor immunotolerance using small molecules or immunostimulatory biological agents such as monoclonal antibody, can inhibit tumor growth and invasion, and subsequently attenuate the tumor metastasis and decrease cancer-caused death.

Asymmetric dimethylarginine (ADMA), a major endogenous inhibitor of nitric oxide synthase, is recently defined as a novel atherogenic factor. Communication via gap junction (GJIC) is involved in the regulation of a variety of endothelial activities, such as cell differentiation and senescence. The aim of this study is to explore the effects of ADMA on connexin43 (Cx43) mediated endothelial GJIC. Lysophosphatidylcholine (LPC) caused the downregulation of Cx43 expression and GJIC dysfunction in cultured human umbilical vein endothelial cells (HUVECs), which were significantly ameliorated by decreasing ADMA accumulation. Furthermore, we found that ADMA (10 micromol x L(-1), 24 h) markedly downregulated Cx43 expression and damaged GJIC function in HUVECs. ADMA also increased production of intracellular reactive oxygen species (ROS) and induced phosphorylation of p38 MAPK. Furthermore, the inhibitory effect of ADMA on Cx43-mediated GJIC could be attenuated by NADPH oxidase inhibitor diphenyleneiodonium and apocynin as well as p38 MAPK inhibitor SB203580, respectively. In conclusion, our present results suggest that ADMA inhibits endothelial GJIC function via downregulating Cx43 expression, which suggesting a novel mechanism linking between elevated ADMA level and progression of atherosclerosis.

B cells are typically characterized by their ability to regulate the immune responses through presenting antigens and producing antibodies. However, a novel B cell subset, named regulatory B cells (Bregs), has been identified. As Tregs, the Bregs are capable of performing both pathogenic and regulatory functions by production of suppressive cytokines, such as IL-10 or TGF-beta1, or by interaction with pathogen T cells or other immune cells. Recent studies indicate that the Bregs play a critical role in the development and resolution of multiple chronic diseases, including inflammatory bowel disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. The identification and the clarification of action mechanisms of the Bregs will greatly contribute to understanding the mechanisms of immune tolerance comprehensively and deeply, and to develop the rational therapeutic strategies for arthritis, diabetes, multiple sclerosis, infectious diseases and cancer, etc. In this review, we summarized the recent insights of identification, characterizations, development, and regulation mechanisms of Bregs and these cells' contribution to the pathogenesis of inflammatory diseases.

Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis., Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8(+) T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice., Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating a life-threatening metastasis.

Objective: To analyze Helicobacter pylori infection in the gastric mucosa of patients with HIV/AIDS in different clinical stages., Methods: This study involved 170 patients with HIV/AIDS and 34 HIV-negative patients. All the patients underwent upper endoscopy and antral gastric biopsy to determine the status of Helicobacter pylori infection using aniline red staining and rapid urease test. The patients with HIV/AIDS were stratified based on CD4(+)T lymphocyte counts and clinical setting into asymptomatic HIV infection (A1, A2) group, symptomatic HIV infection (B1, B2) group and AIDS (A3, B3, C1-3) group., Results: The prevalence of Helicobacter pylori infection in HIV/AIDS patients was 16.5% (28/170), and in the 3 groups classified, the infection rates were 23.4% (11/47), 14.0% (8/57), and 13.6% (9/66), respectively; the infection rate was 47.1% (16/34) in the control group. Helicobacter pylori infection rate in the gastric mucosa of the patients with HIV/AIDS in different clinical stages was significantly lower than that of the control group (P<0.05); the infection rates in symptomatic HIV-infected (B1, B2) group and AIDS (A3, B3, C1-3) group were significantly lower than that in asymptomatic HIV-infected (A1, A2) group (P<0.05)., Conclusion: The low Helicobacter pylori infection rate in HIV/AIDS patients may result from severe immunodeficiency in the gastric mucosa.

Molecules of damage-associated molecular patterns (DAMPs) are a class of substances released to intercellular space or peripheral blood by tissues or cells which are stimulated by insults, ischemia or stress. DAMP molecules can be recognized by Toll like receptors, Nod1-like receptors, or Rig-I like receptors and induce autoimmunity or immune tolerance, which play critical roles in various chronic diseases such as arthritis, atherosclerosis, cancer and systemic lupus erythematosus. DAMP molecules include high-mobility group B protein 1, heat shock proteins and S100 proteins etc. The identification of DAMP molecules and clarification of mechanisms of their action will greatly contribute to reveal the pathological mechanisms of chronic diseases and provide a great opportunity to develop the new strategies for the diagnosis, prevention and treatment for these diseases.

Background: Nonalcoholic Fatty Liver Disease (NAFLD) encompasses a histopathological spectrum of clinical conditions such as simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), and a variant that has degrees of fibrosis. Tumor Necrosis Factor-alpha (TNF-alphalpha) is considered essential for NAFLD. Therefore, we investigated the correlation between TNF-alphalpha gene promoter polymorphism and NAFLD in this human study., Patients and Methods: The TNF-alpha gene polymorphisms at position -238 and -308 were analyzed in 189 Chinese patients with NAFLD and 138 healthy controls by using polymerase chain reaction and restriction fragment length polymorphism assay. The serum levels of TNF-alpha in both patient and control groups were measured by ELISA. The associations of TNF-alpha polymorphism and serum TNF-alpha, and/or insulin resistance, and/or clinical features were analyzed., Results: The carrier frequencies of TNF-alpha gene polymorphism with G/A mutation at -238 were significantly higher in the patients with NAFLD than those in the control subjects (p < 0.05). However, there were no significant differences between the NAFLD patients and control subjects in the polymorphisms at -308 (p > 0.05). In addition, the serum level of TNF-alpha was markedly higher in the patients with NAFLD than in the control subjects (p < 0.05). There were significant associations between TNF-alpha gene polymorphism in the -238 A allele and increased serum TNF-alpha, insulin resistance, as well as increased body mass index in the NAFLD patients., Conclusions: The results indicate that the TNF-alpha gene polymorphism at position -238 is associated with susceptibility of nonalcoholic Fatty Liver Disease in a Chinese population.

Pulmonary fibrosis is a consequence of chronic lung injury and is associated with a high mortality. Despite the pathogenesis of pulmonary fibrosis remaining as an enigma, immune responses play a critical role in the deregulation of wound healing process after lung injury, which leads to fibrosis. Accumulating evidence argues the rationales for current treatments of pulmonary fibrosis using immunosuppressive agents such as corticosteroids. In this study, we report that bleomycin (BLM), a well-known fibrogenic agent functioning as a TLR2 agonist, induced the maturation of dendritic cells and release of cytokines. The BLM activation of TLR2 mediated a time-dependent alteration of immune responses in the lung. These responses resulted in an increase in the tissue-infiltrating proinflammatory cells and cytokines in the early period initially following BLM exposure and an increase in the tissue-infiltrating suppressive immune cells and factors during the later period following BLM exposure. TLR2 deficiency, however, reduced pulmonary inflammation, injury, and subsequently attenuated pulmonary fibrosis. Targeting TLR2 by a TLR2-neutralizing Ab not only markedly decreased animal death but also protected animals from the development of pulmonary fibrosis and reversed the established pulmonary fibrosis through regulating BLM-induced immunosuppressive microenvironments. Our studies suggest that TLR2 is a promising target for the development of therapeutic agents against pulmonary fibrosis and that eliminating immunosuppressive cells and factors via immunostimulants is a novel strategy for fibro-proliferative diseases. Moreover, combining BLM with an anti-TLR2 Ab or TLR2 antagonist for cancer therapy will improve the BLM therapeutic profile by enhancing anti-cancer efficacy and reducing systemic inflammation and pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a relentlessly progressive and typically fatal interstitial lung disease that harms human life severely. Half of patients diagnosed as idiopathic pulmonary fibrosis live no more than three years. No therapy has been clearly shown to prolong survival. Multiple new targets for pulmonary fibrosis have been indicated based on the researches that uncover the molecular and cellular mechanisms for fibrogenic diseases. In this review, we will summarize the clinical treatment for pulmonary fibrogenic diseases and new drugs with clinic trail, and then review focally the prospects of new drugs for pulmonary fibrogenic diseases that target alveolar epithelial cells or myofibroblast, inhibit the angiogenesis, regulate the balance of TH1/TH2 cytokines or block oxide stress.

Hypertension-induced cardiovascular hypertrophy and fibrosis are critical in the development of heart failure. The activity of TLRs has been found to be involved in the development of pressure overload-induced myocardial hypertrophy and cardiac fibrosis. We wondered whether vaccine bacillus Calmette-Guérin (BCG), which activated TLR4 to elicit immune responses, modulated the pressure overload-stimulated cardiovascular hypertrophy and cardiac fibrosis in the murine models of abdominal aortic constriction (AAC)-induced hypertension. Before or after AAC, animals received BCG, TLR4 agonist, IFN-gamma, or TLR4 antagonist i.p. BCG and TLR4 agonist significantly prevented AAC-induced cardiovascular hypertrophy and reactive cardiac fibrosis with no changes in hemodynamics. Moreover, TLR4 antagonist reversed the BCG- and TLR4 agonist-induced actions of anti-cardiovascular hypertrophy and cardiac fibrosis. BCG decreased the expression of TLR2 or TLR4 on the heart tissue but TLR4 agonist increased the expression of TLR2 or TLR4 on the immune cells that infiltrate into the heart tissue. This led to an increased expression ratio of IFN-gamma/TGF-beta in the heart. The cardiac protective effects of BCG and TLR4 agonist are related to their regulation of ERK-Akt and p38-NF-kappaB signal pathways in the heart. In conclusion, the activity of TLR4 plays a critical role in the mediation of pressure overload-induced myocardial hypertrophy and fibrosis. The regulation of immune responses by BCG and TLR4 agonist has a great potential for the prevention and treatment of hypertension-induced myocardial hypertrophy and cardiac fibrosis.

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free beta-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the beta-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor alpha1 (Cbfalpha1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfalpha1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.

Toll-like receptors (TLRs) are widely expressed in the innate and adaptive immune system. They initiate host defense against endogenous and exogenous pathogens containing conserved pathogen associated molecular patterns. TLRs are critical bridges between the innate and adaptive immunity, especially the cellular immunity mediated by T cells. Emerging evidence indicated that B cells express almost all subtypes of TLRs. TLRs play critical role not only in regulation of proliferation, maturation and function of B cells, but also in pathogenesis of diseases, such as systemic lupus erythematosus and chronic lymphocytic leukemia. Targeting TLRs of B cells is a promising therapeutic strategy for these disorders.

Altered peptide ligand (APL), a short peptide with immune regulatory activity and substitutions of a single or multiple amino acids in an antigenic peptide, has shown potential therapeutic effect on autoimmune disease, tumor and virus infection. APL regulates immune responses by interfering the interaction between the major histocompatibility complex (MHC), antigenic peptide and T cell receptor (TCR), or by regulating the intracellular signaling of antigen presenting cells, bystander suppression and inducing heterogenous immune responses. High-specific and high-affinity APL screened from peptide laboratory by phage display, has a potential to be a new resource for drug with antigen specificity.

Although probucol is known to prevent restenosis by regulating vascular remodeling after percutaneous transluminal coronary angioplasty, the mechanisms remain unclear. The present study was designed to investigate whether probucol mediates vascular remodeling via the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. A rabbit restenosis model was used, in which the New Zealand white rabbits received angioplasty with a 3.5 F angioplasty balloon catheter and the proliferation and migration of smooth muscle cells (SMCs) was induced by oxidized low-density lipoprotein (ox-LDL). We evaluated several vascular remodeling parameters and found that probucol prevented lumen restenosis and mediated expansive remodeling with a remodeling index greater than 1 and that the proliferation and migration of SMCs was inhibited. Based on Western blot analyses, probucol decreased the expression of phospho-mitogen-activated protein kinase kinases 1 (p-MEK1) and phospho-ERK1/2 and enhanced the expression of mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and caveolin-1. Cells treated with the MEK1 inhibitor PD98059 demonstrated a remarkable suppression of the effects of probucol. Furthermore, immunofluorescence analysis showed that probucol inhibited the activation of ERK1/2 by preventing its translocation to the nucleus. It was also found that c-myc expression in aortic tissue after angioplasty and the activator protein 1 (AP1) activity in SMCs induced by ox-LDL were decreased with probucol treatment. In conclusion, probucol mediated vascular remodeling to prevent restenosis after angioplasty by down-regulating the ERK1/2 signaling pathway.

Objective: To investigate the relationship among serum leptin, homeostasis model assessment of insulin resistance (HOMA-IR), body mass index (BMI) and dyslipidema in patients with nonalcoholic fatty liver disease (NAFLD)., Methods: Eighty-two patients with NAFLD were divided into mild, moderate and severe NAFLD groups according histological examination results of the liver. Twenty healthy volunteers were chosen as the normal control (NC) group. Fasting insulin, glucose, leptin and lipid levels were measured in the 82 patients with NAFLD and the BMI calculated. IR index of the patients was calculated according to the HOMA method., Results: Leptin, HOMA-IR index, BMI and dyslipidemia showed significant differences between NAFLD and NC groups (P<0.05 or 0.01). Leptin and HOMA-IR index increased with the exacerbation of NAFLD, both of which were positively correlated with the severity of NAFLD., Conclusion: Increased leptin level, HOMA-IR, BMI and dyslipidemia can be important risk factors of NAFLD, and serum leptin level and HOMA-IR are positively correlated with the severity of NAFLD.

Objective: To investigate the mechanism of senescence delay of human diploid fibroblast (2BS) and protecting telomere length by epimedium flavonoids (EF)., Methods: The drug sera of EF were used to treat the 2BS. The population doublings of 2BS cells were observed, the mRNA expression of p16 gene were determined by fluorescence real-time quantitative RT-PCR, the telomerase activation of 2BS cells were determined by TRAP-Hyb, the total retinoblastoma (Rb) and phosphorated Rb protein content were detected by ELISA, the telomere length of 2BS cells were determined by telomere restriction fragment (TRF) Southern blot assay., Results: EF could significantly extend the population doublings of 2BS cells, the expression of p16 mRNA was decreased and the content of phosphorated Rb protein were increased by EF. The telomere lengthening of 2BS cells were improved by EF, but the telomerase was not activated., Conclusion: In senescence human fibroblasts 2BS cells, p16 gene mRNA expression increased, content of phosphorated Rb protein decreased and the telomere length of 2BS shortened, EF might delay the aging of cells through inhibiting the p16 gene expression, promoting the production of phosphorated Rb protein and to protect the length of telomere, but not activating the telomerase.

Phase-contrast X-ray diffraction imaging and high-angular-resolution diffraction combined with phase-contrast radiographic imaging were employed to characterize defects and perfection of a uniformly grown tetragonal lysozyme crystal in the symmetric Laue case. The full-width at half-maximum (FWHM) of a 4 4 0 rocking curve measured from the original crystal was approximately 16.7 arcsec and imperfections including line defects, inclusions and other microdefects were observed in the diffraction images of the crystal. The observed line defects carry distinct dislocation features running approximately along the <1 1 0> growth front and have been found to originate mostly in a central growth area and occasionally in outer growth regions. Inclusions of impurities or formations of foreign particles in the central growth region are resolved in the images with high sensitivity to defects. Slow dehydration led to the broadening of a fairly symmetric 4 4 0 rocking curve by a factor of approximately 2.6, which was primarily attributed to the dehydration-induced microscopic effects that are clearly shown in X-ray diffraction images. The details of the observed defects and the significant change in the revealed microstructures with drying provide insight into the nature of imperfections, nucleation and growth, and the properties of protein crystals.

Surface x-ray diffraction measurements were performed on (111) growth faces of crystals of the cellular iron-storage protein, horse spleen ferritin. Crystal truncation rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48+/-4.5 A and a top layer spacing contraction of 3.9+/-1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

In vascular smooth muscle, increased expression of cyclooxygenase-2 (COX-2) has emerged as an important mechanism for regulation of prostanoid synthesis influenced by vessel injury, cytokines, and growth factors. We have investigated how COX-2 participates in angiotensin II (ANG II)-mediated cell responses in cultured human vascular smooth muscle cells (VSMCs). ANG II type 1 (AT1) receptors induce increased accumulation of COX-2, both at the mRNA and protein levels. ANG II increased transcription of the COX-2 gene; also, nuclear extracts from stimulated cells had increased NF-kappa B binding to its DNA consensus sequence. ANG II-induced COX-2 expression was markedly blunted by inhibition of mitogen-activated protein kinase. Furthermore, the ANG II-induced increase in COX-2 protein abundance was attenuated by both the peroxisome proliferator-activated receptor alpha (PPARalpha) activator Wy-14,643 [pyrinixic acid; 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl) thioacetic acid] and the PPARgamma activator 15d-PGJ2 (15-deoxy-Delta(12-14)-prostaglandin J2). Not only did ANG II increase COX-2 expression and prostaglandin synthesis, ANG II-stimulated DNA synthesis and cell migration were dependent on COX-2 activity. PPARalpha and PPARgamma activators inhibited ANG II-stimulated DNA synthesis and cell migration. These results suggest that ANG II enhances COX-2 expression at the transcription level; also, COX-2 activity plays an important role in mediating ANG II- induced proliferation and migration of VSMCs, suggesting the possibility of magnification of ANG II effects over time due to the induction of COX-2 expression. These results also demonstrate that both the alpha and gamma type of PPAR activators inhibit COX-2 expression induced by angiotensin II in VSMCs which may have therapeutic significance in vascular diseases.

Conventional x-ray diffraction topography is currently used to map defects in the bulk of protein crystals, but the lack of sufficient contrast is frequently a limiting factor. We experimentally demonstrate that this barrier can be circumvented using a method that combines phase sensitive and diffraction imaging principles. Details of defects revealed in tetragonal lysozyme and cubic ferritin crystals are presented and discussed. The approach enabling the detection of the phase changes of diffracted x rays should prove to be useful in the study of defect structures in a broad range of biological macromolecular crystals.

We report a new strategy for the generation of human anticolon cancer monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs derived from peripheral blood mononuclear cells (PBMC) that were transformed by EBV. The immortalized B cells secreting tumor-specific antibodies were identified by HRT-18 cell ELISA and cloned by limiting dilution. Heavy- and light-chain VH-CH1 (gamma) and V kappa-C kappa cDNAs were rescued from ELISA-positive cells wells by RT-PCR. VH and V kappa were amplified by 2nd PCR and linked them together by 3rd PCR assembly with the use of a (Gly4Ser)3 linker. The ScFv cDNAs were then cloned into the fUSE 5 vector and displayed on phage. Phage clones were selected on HRT-18 cells and normal human PBMC. ELISA tested phage clones randomly picked after each panning step. > 80% of the clones showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting anticolon cancer antibodies. This approach offers an effective method by combining in vitro immunization, B-cell expansion for enrichment of specific B-lymphocytes, PCR gene cloning and phage display to make high-affinity human anticolon cancer monoclonal antibody molecules.

Double-axis multiple-crystal X-ray topography, rocking-curve measurements and triple-axis reciprocal-space mapping have been combined to characterize protein crystals using a laboratory source. Crystals of lysozyme and lysozyme crystals doped with acetylated lysozyme impurities were examined. It was shown that the incorporation of acetylated lysozyme into crystals of lysozyme induces mosaic domains that are responsible for the broadening and/or splitting of rocking curves and diffraction-space maps along the direction normal to the reciprocal-lattice vector, while the overall elastic lattice strain of the impurity-doped crystals does not appear to be appreciable in high angular resolution reciprocal-space maps. Multiple-crystal monochromatic X-ray topography, which is highly sensitive to lattice distortions, was used to reveal the spatial distribution of mosaic domains in crystals which correlates with the diffraction features in reciprocal space. Discussions of the influence of acetylated lysozyme on crystal perfection are given in terms of our observations.

Background: Smooth muscle cells in vascular tissue, like tissue within the urogenital sinus, undergo growth and proliferation., Methods: This review attempts to compare and contrast the mechanisms and controlling factors involved in prostatic and vascular tissue. There is a particular focus on the role of catecholamines and alpha-adrenoceptors (alpha-ARs), and on the effects of alpha(1)-AR antagonists (blockers) on cellular dynamics. RESULTS AND CONCLUSIONS The situation in vascular tissue appears analagous to that in prostatic tissue. Certain AR-antagonists, in addition to altering smooth muscle contraction, may have other actions on cellular dynamics., (Copyright 2000 Wiley-Liss Inc.)

Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found previously that alpha1-adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle cells; these effects were attenuated by the tyrosine protein kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of alpha1 receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by alpha1 receptor antagonists. A membrane-permeable Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular Ca2+ plays a critical role in alpha1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, the results suggest that two of them are PLCgamma1 and adapter protein Shc. Also, alpha1 receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44(ERK1/2). The alpha1 receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and intracellular Ca2+ chelator [1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that phosphorylation of tyrosine proteins and intracellular Ca2+ plays a critical role in alpha1 receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.

alpha1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+ ([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by alpha1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the alpha1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fos expression by alpha1A and alpha1B receptors. Brief (1-min) stimulation of alpha1A and alpha1B receptors with PE did not maximally induce c-fos expression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by alpha1A and alpha1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that alpha1-adrenergic receptor-induced c-fos expression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.

Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

Activation of alpha1 adrenergic receptors not only stimulates smooth muscle contraction but also modifies gene expression. We wondered if alpha1 adrenergic receptors could activate transcription of genes regulated by the cAMP response element-binding protein (CREB). Using Rat1 cells stably transfected with each of the three cloned human alpha1 adrenergic receptor subtypes, norepinephrine strongly stimulated CREB phosphorylation in alpha1A and alpha1B but more weakly in alpha1D-transfected cells. Norepinephrine increased the activity of a somatostatin cAMP-regulated enhancer-chloramphenicol acetyltransferase reporter in these cells. alpha1 adrenergic receptors are known to activate protein kinase C (PKC) and increase [Ca2+ ]i. Nonetheless, neither GF109203X, a PKC inhibitor, nor BAPTA-AM, a calcium chelator, blocked phosphorylation of CREB induced by norepinephrine. In addition, alpha1 adrenergic receptor-induced CREB phosphorylation was not mediated via the mitogen-activated protein kinase pathway because norepinephrine did not stimulate mitogen-activated protein kinase activity in these cells. Activation of alpha1 adrenergic receptors increased cAMP accumulation in these cells. Norepinephrine-induced cAMP-regulated enhancer-chloramphenicol acetyltransferase activity was inhibited either by expression of the PKA inhibitory peptide or a dominant negative PKA regulatory subunit mutant. These results demonstrate that alpha1 adrenergic receptors activate the transcription factor CREB by a PKA-dependent pathway.

Proliferation and migration of vascular smooth-muscle cells (VSMCs), stimulated by a variety of growth factors, play a critical role in the pathogenesis of vascular diseases. We found unexpectedly that doxazosin, an alpha1-adrenergic-receptor antagonist, inhibits serum-stimulated proliferation of cultured human VSMCs. Subsequent experiments systematically investigated inhibitory effects of doxazosin on mitogenesis stimulated in VSMCs by platelet-derived growth factor (PDGF), epidermal growth factor, and G protein-coupled receptor agonists thrombin and angiotensin II. Doxazosin attenuated the stimulation of DNA synthesis for each of these ligands with median inhibitory concentrations (IC50s) from 0.3 to 1 microM. PDGF-AB (1 nM) increased cell number; doxazosin inhibited this response by 70-80%. Prazosin, a related alpha1-receptor antagonist, had similar but less potent effects on inhibiting mitogenesis in these cells. Doxazosin and prazosin inhibited PDGF-AB-stimulated and insulin-like growth factor (IGF-I)-stimulated migration of VSMCs by approximately 40-50%. These effects of doxazosin were likely unrelated to alpha1-receptor blockade because pretreatment of cells with phenoxybenzamine, an irreversible alpha1 antagonist, did not change the capacity of doxazosin to inhibit of PDGF-stimulated mitogenesis. Also, doxazosin inhibited PDGF-stimulated DNA synthesis in NIH 3T3 cells, which do not express alpha1 receptors. These results suggest that doxazosin is a potent inhibitor of VSMC proliferation and migration through a mechanism unrelated to alpha1-receptor antagonism.

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.

Hyperinsulinemia has been implicated as an important risk factor for the development of accelerated cardiovascular disease. We wondered if insulin or IGF-I induced expression of alpha1 adrenergic receptors in vascular smooth muscle cells (VSMCs) which could enhance smooth muscle contraction and cell growth activated by catecholamines. Rat aortic VSMCs were incubated with insulin or IGF-I for various times and expression of alpha1 receptors was detected using [3H]prazosin binding. Both insulin and IGF-I increased alpha1 receptor number; also, these peptides increased expression of the alpha1D receptor gene with no change in expression of the alpha1B receptor gene as detected by RNase protection assays. Using Western blotting, we found that these peptides increased expression of the alpha1D receptor subtype in these cells. Increased expression of the alpha1D receptor mRNA was inhibited by the receptor tyrosine kinase inhibitor genistein and the PI 3-kinase inhibitor wortmannin but was not inhibited by protein kinase C inhibitor H7 or the L-type calcium channel blocker nifedipine. Preincubation of cells with insulin or IGF-I enhanced subsequent norepinephrine stimulation of mitogen activated kinase activity. These results suggest that insulin/IGF-I regulate expression of alpha1 receptors in VSMCs and potentially enhance the effects of catecholamines in settings of hyperinsulinemia.

The capacity of various growth factors to induce c-fos expression is diminished with senescence. Because adenosine 3',5'-cyclic monophosphate (cAMP)-mediated responses are also blunted with aging, we wondered whether cAMP-induced c-fos gene expression might be impaired with senescence. Using IMR fibroblasts, we found that prostaglandin E1 (PGE1) and forskolin, stimulators of cAMP accumulation in young and senescent cells, increased abundance of c-fos and junB mRNA more in young than senescent cells. The abundance of the cAMP response element binding protein (CREB), a transcription factor which enhances gene expression when phosphorylated by protein kinase A, was markedly decreased in both whole cell and nuclear extracts of senescent cells, in both Western blotting and in gel retardation assays. Also, PGE1-induced phosphorylation of CREB by protein kinase A was markedly attenuated in senescent cells. There is a marked decrement in expression of CREB with senescence, and the results suggest the possibility that the diminished expression of CREB may contribute to altered cAMP-mediated regulation of gene expression with senescence.

Alpha 1 adrenoreceptors mediate inotropic and chronotropic responses in the heart and vaso-constriction in blood vessels. Pharmacological studies using selective antagonists for subtypes of alpha 1 receptor suggest that different receptor subtypes may mediate different physiological effects. Prior studies have demonstrated that alpha 1-mediated cardiac responses are altered in aging and that alpha 1 receptor number declines with age in the rat heart. The distribution of alpha 1 receptor subtypes in cardiac tissue and subtype-specific changes with aging, however, have not been established. Using R Nase protection assays and in situ hybridization techniques, we have detected message for alpha 1B, alpha 1A, and alpha 1D in all four chambers of the rat heart and in multiple blood vessels. We found no significant changes in alpha 1 receptor subtype mRNA levels in hearts from young (4 months) and old (25 months) Sprague-Dawley rats. Also, there was expression of these three subtypes in all blood vessels assayed. We conclude that transcriptional regulation of alpha 1 adrenoreceptor subtypes does not account for age-related changes in cardiac alpha-adrenergic responsiveness.

Induction of heat shock proteins (hsp) most likely is a homeostatic mechanism in response to metabolic and environmental insults. We have investigated signal transduction mechanisms involved in alpha1, adrenergic receptor stimulation of hsp7O gene expression in isolated aortas with age. We found that alpha1 adrenergic agonists directly induced hsp70 mRNA in rat aorta in vitro; the alpha1, selective antagonist prazosin blocked this effect whereas chloroethylclonidine, an antagonist which has some selectivity for alpha1B receptors, was ineffective. This response was insensitive to pertussis toxin and was partially blocked by the protein kinase C inhibitor H7. Removal of extracellular calcium attenuated induction of hsp70 mRNA but not the induction of c-fos or c-myc. The induction of hsp70 mRNA by either norepinephrine or by phorbol dibutyrate was blunted in aortas from old (24-27 mo) rats whereas c-fos responses were not diminished in the older vessels. The hsp70 response to elevated temperature (42 degrees C) was not changed with age. Activation of hsp70 expression most likely involves a pertussis toxin insensitive G protein which activates protein kinase C, and requires extracellular calcium. With age, hsp70 gene expression induced by stimulation of alpha1 adrenergic receptors is markedly attenuated, which could modify responses to stress or vascular injury with aging.

Activation of alpha1 adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of alpha1 receptors may induce mitogenesis in HVSMCs, we have found that alpha1 receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of alpha1 receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85 alpha subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a beta adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of alpha1 receptors and by pertussis toxin, suggesting that alpha1 receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca2+ L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of alpha1 receptors activates PI 3-kinase in HVSMCs and that alpha1 receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to alpha1 receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.

The role of the endothelium in modulating smooth muscle cell growth is unclear. alpha 1 adrenergic receptors activate proto-oncogene expression in smooth muscle. We have now found in rat aorta that carbachol, a muscarinic cholinergic agonist that promotes release of nitric oxide (NO), inhibits expression of c-fos and c-jun mRNA induced by alpha 1 receptors. NO synthase inhibitors blocked the effects of carbachol on c-fos mRNA and a cGMP analog mimicked carbachol. After balloon injury in rat aorta using in situ hybridization, the catecholamine-induced increase in c-fos mRNA expression in the medial layer was inhibited by the alpha 1 receptor antagonists, prazosin and chloroethylclonidine. In the neointima, this response was fully blocked by prazosin; however, chloroethylclonidine only partially inhibited it. These results suggest that NO, acting through a cGMP-dependent mechanism, inhibits expression of the c-fos and c-jun genes in arteries, which may contribute to the growth-inhibiting effects of the endothelium. After endothelial damage, the activation of c-fos in neointima by adrenergic stimulation may involve a subtype of alpha 1 receptor different from that utilized in medial smooth muscle.

We recently reported that alpha1-adrenoceptor agonists, administered at the beginning of neurulation (Stage 11a) in rat embryos grown in culture, interfere with normal development of the left/right body axis leading to situs inversus. Despite these pharmacological findings, expression of alpha1-adrenoceptor genes at such an early stage of development has not been demonstrated. In the present study, we examined the expression of mRNAs for cloned alpha1-adrenoceptor subtypes in rat embryos at Stage 11a. Timed-pregnant Sprague-Dawley rats were killed in the morning of gestational day 9 (vaginal plug day = day 0), and the implantation sites were removed. The implantation sites were separated into embryo, ectoplacental cone and decidua, only those at Stage 11a were selected, and these were immediately frozen on dry ice, and subsequently their total RNA was isolated. RNase protection assays were then performed for cloned alpha1a-, alpha 1b- and alpha1d-adrenocepter subtypes using 20-30 mug of total RNA for each hybridization. In all tissues, strong and weak signals were detected for alpha1b- and alpha1a-adrenoceptor subtype mRNAs, respectively. In contrast, a signal for alpha1d mRNA was not detected in any tissues. These results, together with previous pharmacological findings, suggest the existence of alpha1a- and alpha1b-adrenoceptor subtypes in rat embryos at Stage 11a.

Alterations in adenylyl cyclase activity in cultured cells after prolonged exposure to drugs such as morphine have been extensively studied as models for drug tolerance and withdrawal. NG108-15 cells develop increased intracellular cAMP concentrations after abrupt withdrawal from chronic treatment with the muscarinic cholinergic agonist carbachol. To determine whether this withdrawal-induced increase in cAMP modifies gene expression, we studied phosphorylation of the cAMP response element-binding protein (CREB) and expression of the c-fos gene, known to contain a cAMP response element, in NG108-15 cells after abrupt withdrawal from chronic treatment with carbachol. Prostaglandin E1, which activates adenylyl cyclase, caused concentration-dependent increases in the phosphorylation of CREB and in the abundance of c-fos mRNA. These changes occurred with small increments in cAMP accumulation. In cells treated with carbachol for 48 hr, induction of withdrawal with the muscarinic antagonist atropine led to a small increase in intracellular cAMP concentration but an 11.6-fold increase in the phosphorylation of CREB and a 3.4-fold increase in accumulation of c-fos mRNA. The adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, which attenuated the chronic carbachol-induced increase in cAMP concentration, prevented the increased phosphorylation of CREB and the enhanced accumulation of c-fos mRNA during atropine-induced withdrawal. These results indicate that expression of the c-fos gene is induced by the small increments in cAMP concentration that can occur in cells on withdrawal from chronic treatment with drugs such as muscarinic agonists.