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RT @dearjerksmusic: Hey hey here I am on Illinois radio this morning (the "running late" part is a mystery to me though cuz I was on hold t… 1 day ago
At least, that’s this writer’s take on it. If you look at what the Left and Right stand for, it seems pretty clear. If you listen to how they speak, it seems even more so. And when you look at how they behave, it seems irrefutable.
“Conservatives” are afraid of losing elections, so they rig elections and take away non-rich-white males’ voting rights.
“Conservatives” are afraid of their own government, so they buy themselves more guns than any person could ever use.
“Conservatives” are afraid of Ebola, so they want to force medical workers into solitary confinement just because they are scared.
On and on, and it never stops. “Republicans” are a bunch of wimps who, when it comes down to Fight or Flight, chose Flight 100% of the time. Big-talking wussies like Chris Christie and John McCain, who run and hide, cowering in the shadows if ever they are called on their braggadocio.
Liberals behave, of course, in the exact opposite manner. They confront problems and deal with them. They are “Fighters”, not “Flighters”.
Next Tuesday, you have a choice: give control of the most powerful nation in the world to a load of scaredy-cat, bullying little wankers (Repubs), or a bunch of flawed but courageous Liberals (Democrats). It’s kind of a big deal, Gentle Reader. A pretty big and very consequential choice.
Back in the 70’s, a truly epic bit of criminal activity was committed and then covered up by the Nixon administration. The crime and the cover up both got people ticked off, and Nixon got ridden out of office on a rail. Deservedly so.
In the 90’s, a jackass Prexy was boinking an intern, which was not a crime in and of itself. The cover up was, however, and B. J. Clinton barely survived the thrashing he so richly deserved.
In the early Oughts, the Bushies leaked the name of one of our clandestine agents in Iran. None of the scumbucket traitors who committed this crime got the punishment they deserved. Many innocent Iranians did get punishment that they did not deserve.
Many conspiracy theorists are convinced that the government is keeping secrets about UFOs, 9/11, and other such things. There is no punishment for being silly.
Not all of these secrets are equally worthy of being revealed. When we let the sunshine in, we need to make sure are illuminating the deserving, and keep the innocent from becoming collateral damage. Example: Steubenville, Ohio, where an attempt to cover up a ring of gambling, sex crimes and corruption has now thrust both the innocent and the guilty into the spotlight.
An association between unrecognized gastroesophageal reflux disease and excessive daytime sleepiness in Taiwanese subjects suspected to have liver disease: a pilot study | BMC Gastroenterology | Full Text
An association between unrecognized gastroesophageal reflux disease and excessive daytime sleepiness in Taiwanese subjects suspected to have liver disease: a pilot study
In traditional Chinese culture, liver disease is believed to underlie excessive daytime sleepiness (EDS). Consequently, Chinese patients with complaints of EDS and physicians who treat them suspect that a liver abnormality is present. If liver disease is ruled out, these patients are often discharged without treatment. Gastroesophageal reflux disease (GERD) is a common disorder also associated with EDS. This pilot study was undertaken to determine the prevalence of GERD among Taiwanese patients with complaints of EDS suspected to be related to liver disease but in whom no evidence for the latter was found.
From July 2009 to December 2009, 121 outpatients who presented to or were referred to the Department of Gastroenterology and Hepatology of the Chiayi Gung Memorial Hospital for evaluation of a complaint of EDS thought to be due to liver disease were examined. Demographic data were collected, and physical examinations and liver function tests were performed. Forty-eight patients had liver disease and were excluded. The Chinese Epworth Sleepiness Scale questionnaire (Chinese ESS) and the Chinese Gastroesophageal Reflux Disease Questionnaire (CGERDQ) were then administered to 73 included patients.
More than half (56.2%) of the included patients were found to suffer from GERD. Patients with symptoms of GERD had higher mean CGERDQ scores than patients without symptoms of the disorder (18.88 ± 5.49 and 5.56 ± 3.57, respectively; P < 0.001). Patients with symptoms of GERD also had higher mean Chinese ESS scores than patients without symptoms (8.80 ± 5.49 and 3.13 ± 3.50, respectively; P < 0.001). Chinese ESS scores indicative of EDS were observed in 48.8% of patients with symptoms of GERD and in 3.1% of those without symptoms (P < 0.001). Differences between the two groups retained their significance after controlling for potential confounders.
A significant percentage of Taiwanese patients who complained of EDS and were admitted to our Hepatology/Gastroenterology Department due to a suspicion of liver disease actually had symptoms of GERD. Further studies are needed to ascertain whether treatment of GERD will effectively resolve EDS in these patients.
Gastroesophageal reflux disease (GERD) is a common disorder affecting approximately 10-38% of the adult population in Western countries and an estimated 17% of adults living in Xi'an, Shaanxi province in China [1–3]. Consequences of GERD include, but are not limited to, Barrett's esophagus and adenocarcinoma of the esophagus. In addition, GERD is associated with upper respiratory disease, adversely affects quality of life (QOL), and represents a significant economic consideration in terms of treatment costs [1].
Sleepiness, or somnolence, is a complex state influenced by factors such as quantity and quality of prior sleep, circadian time, medications, and environmental stimuli as well as by medical, neurological, emotional, and psychiatric conditions. When sleep is not desired, somnolence is unwanted. Continuous inadequate sleep negatively impacts cognitive function, physical performance, overall well-being, and QOL [4]. The pathologic or inappropriate occurrence of somnolence is clinically referred to as excessive daytime sleepiness (EDS) [5].
Sleep contributes to a reduction in normal anti-reflux mechanisms; rates of swallowing and salivation are decreased, pressure from the upper and lower esophageal sphincters is decreased, gastric emptying is delayed, and the "heartburn-signal" is depressed. The supine position of most sleepers is thought to facilitate gastroesophageal reflux, a major cause of disrupted sleep due to awakenings from heartburn, dyspepsia, acid brash, coughing, or choking [5, 6]. Not unexpectedly, more than half of patients with chronic GERD report nocturnal symptoms [2–4, 7–10]. The consequences of nocturnal GERD are thought to exceed those of daytime GERD in terms of increased risk of esophageal lesions and respiratory complications, poor QOL, inadequate sleep, EDS, poor work productivity, and increased costs. Like daytime GERD, nocturnal GERD can be managed with surgical therapies or proton pump inhibitors such that heartburn and regurgitation are reduced concurrently with an improvement in sleep.
In addition to GERD, EDS is associated with other co-morbidities including liver disease [11–17]. Daytime fatigue is frequently encountered in patients carrying hepatitis virus and in those with primary biliary cirrhosis, cirrhosis without hepatic encephalopathy, and nonalcoholic fatty liver disease. In traditional Chinese culture, liver disease is believed to underlie sleep disorders. Consequently, Chinese patients with complaints of daytime sleepiness and physicians who treat such patients suspect that a liver abnormality is present. Unfortunately, if liver disease is ruled out, these patients are often discharged without treatment or follow-up examinations.
The present pilot study was undertaken to determine the prevalence of GERD among patients who presented to or were referred to the Department of Gastroenterology and Hepatology with complaints of daytime sleepiness suspected to be related to an underlying liver disease but in whom no evidence for liver disease was obtained.
From July 2009 to December 2009, outpatients who either presented to or were referred to the Department of Gastroenterology and Hepatology of the Chiayi Gung Memorial Hospital for evaluation of a previously uninvestigated sleep disorder were included. In Taiwan, an official referral to a gastroenterology/hepatology department is not required; therefore patients in this study were admitted with or without a referral. Exclusion criteria were: previous diagnosis with any type of sleep disturbance; diagnosis of GERD; current treatment for GERD by a physician; and previous abnormal liver function test findings. Patients known to have chronic hepatitis B or C or to be carriers of hepatitis B or C were also excluded. Written informed consent was obtained from all patients, and this study was approved by the Ethical Committee for Human Clinical Trials of the Chiayi Gung Memorial Hospital, R.O.C. (Trial Number 98-2033C).
Demographic data were collected at baseline, including information regarding use of medications, alcohol, and tobacco. A physical examination was performed, and a blood sample was collected from each patient for measurements of hepatitis B serum antigen (HBsAg), anti-HCV Ab, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Patients with any abnormal measurements were then excluded. None of these patients had a disease history warranting additional tests (such as a complete blood count). Furthermore, additional tests were not permitted by the insurance policies of these patients.
The Chinese Epworth sleepiness scale questionnaire (Chinese ESS) was administered to all included patients by the authors. The Chinese ESS is based on the Epworth sleepiness scale for measurement of daytime sleepiness; the latter is considered to be the most discriminating test for measurement of EDS [18–21]. Designed for diagnosis of EDS in all Chinese subjects, the Chinese ESS has been validated for use in Taiwanese and Mandarin Chinese populations [22]. Specifically, the Chinese ESS measures the propensity for sleep under a variety of life-related situations [21]. The questionnaire consists of eight self-reported questions, with each receiving a score of 0 to 3. Total possible scores range from 0 (the least sleepy) to 24 (the most sleepy), with a total score of 5.9 ± 2.2 considered normal for this group of subjects [19, 20]. A score of 10 is indicative of EDS and is considered the threshold for referral for sleep evaluation [21].
The Chinese Gastroesophageal Reflux Disease Questionnaire (CGERDQ) was also administered to all patients included in the study. This questionnaire, which is designed to identify individuals with symptoms of gastroesophageal reflux, is comprised of eight questions. Each question evaluates the frequency and severity of a symptom of GERD and is scored from 0-3, with a total score ≥12 serving to indicate the presence of GERD [23]. The questionnaire has been validated for use in Chinese subjects and has been shown to be capable of detecting changes in symptoms [23, 24].
Group comparisons for normally distributed continuous variables between subjects with and without GERD symptoms were performed using independent two-sample t tests. The Chi-square test or Fisher's exact test was used for comparisons of categorical variables. Normally distributed continuous data are presented as means ± standard deviation whereas categorical data are represented by numbers and percentage. Stepwise linear regression analysis was performed to identify the factors influencing the Chinese ESS. All statistical assessments were two-sided, with a P value of < 0.05 indicating statistical significance. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS Inc., Chicago, IL, USA).
Between July 2009 and December 2009, 121 consecutive patients with a primary complaint of EDS were initially included. In every case, either the patient and/or the referring practitioner suspected that the complaint was related to liver disease. Of these, 48 patients were subsequently excluded because of the presence of chronic liver disease. Specifically, chronic HBV hepatitis was found in 12 patients (of which 5 also had fatty liver), HCV hepatitis was found in 13 patients (of which 6 also had fatty liver), HBV and HCV hepatitis were found to be present in 1 patient, and fatty liver only was found in the remaining 22 patients. Seventy-three consecutive patients without liver disease who presented to or were referred to the outpatient Hepatology and Gastroenterology Clinic with a primary complaint of EDS were then selected for this study. The baseline characteristics of these 73 subjects are presented in Table 1. This group included 34 (46.6%) males and 39 (53.4%) females ranging in age from 17 to 78 years (mean = 45.75 ± 17.82 years). Based on the results of the CGERDQ, GERD symptoms were present in 41 (56.2%) of the 73 subjects. Mean CGERDQ scores in the GERD and non-GERD groups were 18.88 ± 5.49 and 5.56 ± 3.57, respectively (P < 0.001). The GERD group also contained more patients with positive Chinese ESS scores (>10) as compared to those in the non-GERD group (48.8% versus 3.1%, P < 0.001). The mean Chinese ESS score for subjects with GERD was higher than that for subjects without the disorder (8.80 ± 5.49 versus 3.13 ± 3.50, P < 0.001). Most subjects included in this study did not use alcohol (71.2%), acid suppression (75.3%), or sedatives/hypnotics (95.9%). In addition, 69.9% and 91.8%, respectively, of subjects in this study did not smoke or snore. All subjects were found to be negative for HBsAg and Anti-HCV Ab, and AST and ALT values for all subjects were within the normal range.
Baseline characteristics of 73 patients with or without gastroesophageal reflux disease as determined by the Chinese Gastroesophageal Reflux Disease Questionnaire
P values were determined from aindependent two sample t tests, bChi-square tests, or cFisher's exact test.
Simple and multiple linear regression analyses were performed to identify factors influencing the Chinese ESS. Findings of these analyses are presented in Table 2. The only variable found to influence the Chinese ESS significantly was the CGERDQ score (P < 0.001). After controlling for the potential confounders of age and gender, the CGERDQ score remained significantly and positively associated with the Chinese ESS. The adjusted R 2 was 0.426, indicating that the statistical modeling accounted for approximately 43% of the variation in the Chinese ESS.
In this pilot study, 73 Taiwanese subjects with complaints of EDS but for whom no evidence of liver disease was obtained were examined for the presence of GERD; remarkably, more than half (56.2%) were found to suffer from this disorder. Mean CGERDQ scores for patients with symptoms of GERD (GERD group) were 3.4-fold higher than those for patients without symptoms of the disorder (non-GERD group). Patients in the GERD group were found to have significantly higher mean Chinese ESS scores than those in the non-GERD group. Additionally, more patients in the GERD group had Chinese ESS scores that exceeded 10. The differences in CGERDQ and Chinese ESS scores between the GERD and non-GERD groups retained their significance after controlling for the potential confounders of age and gender. While a cause and effect relationship remains to be established, these findings support an association between GERD and EDS in Taiwanese subjects without liver disease.
The mean ESS score for the entire cohort of patients examined in this study (6.32 ± 5.49) was comparable to that reported for the normal community (5.9 ± 2.2) [19, 20] whereas mean scores for patients in the GERD and non-GERD groups were 8.80 ± 5.49 and 3.13 ± 3.50, respectively. It is unclear why the mean score for the GERD group fell below that considered to indicate EDS and why the mean score for the non-GERD group fell below that for the normal community. It is possible that some of the subjects in this study who claimed to have EDS did not in fact suffer from more daytime fatigue than do members of the local population. Additionally, some patients in this study may have not been overly concerned about their daytime sleepiness and therefore presented directly to a gastroenterologist in preference to a sleep center.
A relationship between GERD and daytime somnolence has been described in other studies. For example, Demeter et al. [4] reported that the severity of GERD as assessed by panendoscopy and characterized by the Savary-Miller classification correlated positively with the degree of daytime somnolence as assessed by the ESS. Specifically, mild somnolence was observed in 29% of GERD 0 patients, 39% of GERD I patients, and 46% of GERD II patients; however 77% of GERD III patients suffered from significant somnolence. Guda et al. [25], who evaluated 385 consecutive subjects at an outpatient clinic for sleep disorders, reported that those with a diagnosis of GERD had significantly higher ESS scores than those diagnosed without GERD. Furthermore, Chen et al. [26] reported that patients with nighttime heartburn complain of significantly greater subjective sleep impairment as determined by the Pittsburgh Sleep Quality Index as compared to those without nighttime heartburn. However, it should be noted that Suurna et al. [27] observed no differences between patients with and without nighttime heartburn in any objective sleep parameter during an overnight polysomnographic study.
In a recently published systematic review of the literature [10], the mean prevalence of nocturnal GERD was reported to be 54%. Farup et al. [3], who conducted a national random-sample telephone survey, reported that 71% of subjects with GERD experienced nocturnal heartburn. In a separate study [28] of 1,000 individuals who experienced heartburn at least once weekly, the prevalence of nocturnal heartburn was found to be 79%; three-quarters of those with nocturnal heartburn revealed that their heartburn episodes negatively impacted their sleep. In the present study, the prevalence of nocturnal as compared to daytime GERD was not directly determined. No patient included in this study complained of nocturnal heartburn but all were troubled by EDS such that the assistance of a gastroenterologist was sought. Based on inquiries conducted after completion of the questionnaires, it was apparent that all subjects in the study were likely to have experienced at least light nocturnal heartburn approximately once weekly. Unfortunately, no accepted questionnaire is currently available to define the degree of nocturnal heartburn, and patients in this study were not willing to submit to measurements of nighttime heartburn with instruments required for esophageal testing. Future studies focusing on consequences of nocturnal as opposed to daytime GERD are indicated; nocturnal symptoms are associated with significant impairment of health-related QOL and nocturnal acid reflux is associated with severe esophagitis, stricture, and esophageal adenocarcinoma [3, 28].
As reviewed by Orr [10], consequences of reflux events include difficulty initiating sleep, arousals from sleep, daytime fatigue, and impaired work productivity. It therefore appears reasonable to consider sleep-related GERD a distinct clinical entity [10]. Fortunately, GERD can be effectively managed using such approaches as head of bed elevation, proton pump inhibitor therapy, H2 receptor antagonist therapy, and Nissen funoplication [5]. According to the ProGERD initiatives [2], the impairment of QOL by GERD can be significantly attenuated or normalized in as little as two weeks by treatment with esomeprazole. In the present study, significantly more patients in the GERD group as compared to the non-GERD group utilized acid suppression (15% as compared to 9.4%, P = 0.007). This finding further supports the hypothesis that GERD contributes to EDS in Taiwanese subjects.
The present study is unique in that subjects were included who complained of EDS rather than symptoms of GERD. Considering the negative impact of EDS, the high prevalence of GERD [1, 2], and the clear association between GERD and fatigue [4, 7], it would appear prudent for general practitioners to consider GERD as an underlying contributor to daytime somnolence in patients with normal liver function. Following a diagnosis of GERD, initiation of appropriate therapy and subsequent evaluation of that therapy would be indicated. In support of these recommendations, pantoprazole was found to provide more significant reductions in reflux symptoms and ESS scores than placebo after administration to patients with mild-moderate obstructive sleep-disordered breathing and GERD [27].
The presence of snoring and sleep apnea are relevant to an evaluation of EDS. In the present study, 14.6% of patients in the GERD group but 0% of patients in the non-GERD group reported snoring. According to patients in the GERD group, snoring was neither frequent nor severe. However, no tests were performed to determine either the accuracy of these assessments or the presence of obstructive sleep apnea in these subjects. Further research is warranted to determine the relative extents to which GERD and sleep apnea contribute to EDS in Taiwanese subjects and other populations.
The most important limitations of this study are the small sample size, the absence of confirmation of a diagnosis of GERD by endoscopy or other appropriate diagnostic test, and the lack of a control group. This pilot study was designed to determine the prevalence with which Taiwanese patients without liver disease but who complain of EDS exhibit symptoms of GERD. Furthermore, endoscopy is not routinely performed in hepatology clinics in Taiwan. Future randomized, controlled clinical studies that include a larger number of patients, an appropriate control group, and tests confirming the presence of GERD are clearly warranted. In addition, the present study did not evaluate QOL or social or psychological factors with the potential to contribute to EDS. Given the demonstrated negative impact of both GERD and daytime somnolence on QOL, such evaluations are also warranted.
EDS is a common complaint of patients with liver disease [12–17]. However, Chinese patients with complaints of EDS but subsequently found to have normal liver function are often left untreated. In these patients, EDS persists and the underlying cause is left undiagnosed. This small pilot study reveals that many patients with complaints of EDS and who were admitted to our Department of Hepatology and Gastroenterology due to a suspicion of liver disease actually had symptoms of GERD. Further studies are needed to ascertain whether appropriate treatment of GERD will effectively resolve EDS in these patients.
JHH designed the study, prepared the protocol, and wrote the first draft of the paper. SWL revised the study design. YYH collected the data. NHC directed and supervised the performance of the study and the writing of the paper. All authors have approved the final draft of this paper.
The Forkhead/Fox transcription factor Foxc2 is a critical regulator of osteogenesis and angiogenesis of cells. Bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and fibroblasts. The present study investigates the role of Foxc2 overexpression in osteogenesis and angiogenesis of BMSCs in vitro.
BMSCs were isolated from SD rat femurs and tibias, and characterized by cell surface antigen identification and osteoblasts and adipocytes differentiation. The cells were transfected with lentiviral Foxc2 (Lv-Foxc2) or green fluorescent protein (Lv-GFP). Seventy hours later, Foxc2 expression was observed using real time-PCR and Western blot. The transfected cells were stained with Alizarin red S or alkaline phosphatase (ALP) after osteogenic induction. Meanwhile, the expression levels of osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF) and platelet-derived growth factor-β (PDGF-β) were measured by real time-PCR, Western blot and immunostaining.
Results of cell characterization showed that the cells were positive to CD44 (99.56%) and negative to CD34 (0.44%), and could differentiate into osteoblasts and adipocytes. Foxc2 overexpression not only increased the numbers of mineralized nodes and ALP activity, but also enhanced the expressions of Runx2, OCN, VEGF and PDGF-β in transfected BMSCs after osteogenic induction. The effects of Foxc2 on osteogenesis and angiogenesis were significantly different between Lv-Foxc2 transfected BMSCs and Lv-GFP transfected BMSCs (P<0.05). In addition, the MAPK-specific inhibitors, PD98059 and LY294002, blocked the Foxc2-induced regulation of BMSC differentiation.
Foxc2 gene is successfully transfected into BMSCs with stable and high expression. The overexpression of Foxc2 acts on BMSCs to stimulate osteogenesis and angiogenesis. The effect of Foxc2 on angiogenesis of the cells is mediated via activating PI3K and ERK.
Bone formation is a temporally controlled, multistep process, and the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts is central in the maintenance of bone integrity [1]. Both osteoblasts and adipocytes share a common progenitor derived from bone marrow mesenchymal stem cells (BMSCs), and bone loss is associated with an expansion of adipose tissue in bone marrow [2]. Angiogenesis is a process involving endothelial cell proliferation and migration, and vascular tube formation; it always accompanies osteoblast differentiation and bone formation [3, 4].
BMSCs have been a focus of research in stem cell-based tissue engineering during the last decade. This is due to their capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and fibroblasts. Studies have shown that BMSCs have the potential to promote angiogenesis, which makes them an ideal cell type in engineering vascularized tissue [5, 6]. The cells can also differentiate into osteoblasts and contribute to bone formation [7–9]. The osteoblast differentiation is mediated by various signaling molecules and transcriptional regulators, such as Wnt/beta-catenin, Notch and Hedgehog signaling pathways, and Runx2 and Osterix transcriptional factors [10, 11].
Foxc2 is a member of the family of winged helix/forkhead transcription factors and is known to be expressed mainly in mesenchymal tissue [12]. Fox protein family members are important for a wide spectrum of biological processes, including metabolism, development, differentiation, proliferation, apoptosis, migration, invasion and longevity [13]. It has been reported that Foxc2-deficient mice display defective formation of the aortic arches, multiple craniofacial bones and vertebral columns, indicating an essential role of the gene in the normal development of the axial skeleton and aortic arches in mice [14, 15]. Besides, Foxc2 inhibits white adipocyte differentiation by suppressing the PPARγ-induced adipogenic gene expression [16], and it regulates angiogenesis by regulating the expressions of various genes involved in the angiogenic process through activating their promoters via Fox-binding elements (FBEs) [17, 18]. In addition, recent studies demonstrate that haplodeficiency of Foxc2 may result in impaired formation of tumor blood vessels as well as reduced tumor growth, and thereby provide evidence for the association of Foxc2 with the metastasis and angiogenesis of tumors [19, 20]. However, it remains unclear how this transcription factor functions during osteogenesis and angiogenesis.
The present study was aimed to determine the role of Foxc2 overexpression on osteogenesis and angiogenesis of BMSCs. The molecular mechanisms of Foxc2 transcriptional regulation were also investigated.
Lentivirus packaging system, including plasmid pGC-FU, pHelper 1.0, pHelper 2.0 and plasmid Foxc2, was purchased from Shanghai GeneChem Co., China. Alizarin red S and Oil Red O staining kits were obtained from Winchem Industrial Co. Ltd., China. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBIC, USA. Antibodies against Foxc2, anti-CD44, anti-CD34 and β-actin were bought from Santa Cruz Biotechnology, USA. Antibodies against OCN, VEGF and PDGF-β were purchased from Abcam, USA. Antibodies against Runx2, ERK and PI3K were purchased from Novus, USA.
Six male SD rats, weighing 150 ± 2 g, were obtained from the Laboratory Animal Center in Medical College of Xi’an Jiaotong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee at Xi’an Jiaotong University. BMSCs were isolated following the method described in [21]. In brief, the femurs and tibias of the rats were removed. Muscles and extraosteal tissues were trimmed. Bone marrow cells were flushed and centrifuged on a 1.073 g/mL Percoll density gradient (Pharmacia, St. Louis, USA). The cells were washed twice with PBS, seeded into 25 cm2 cell culture flasks, and cultivated in L-DMEM supplemented with 10% FBS and 20 mg penicillin-streptomycin/ml. Finally, the cultures were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The medium was changed on the 4th day of culture and every 3 days after that. When the cells became subconfluent, usually after 7–10 days of culture, cells were detached using trypsin/EDTA (0.25% trypsin and 0.02% EDTA) (Sigma), concentrated by centrifugation at 1000 rpm for 5 min and then suspended in medium. An analysis of cell surface molecules, CD44 and CD34, was conducted on passage 3 cultures using flow cytometry. Osteoblasts and adipocytes differentiation was detected with Alizarin red S and Oil Red O staining kits under different induction conditions.
The transfected BMSCs were seeded in 24-well plates at a density of 1×106 cells/well. Osteogenic differentiation was induced under an osteogenic condition [DMEM supplemented with 10% FBS, 0.1 μM dexamethasone (Sigma), 50 μM ascorbate acid (Sigma), and 10 mM β-glycerophosphate sodium (Sigma)] and an adipogenic condition [DMEM supplemented with 10% FBS, 200 μM indomethacin (Sigma), 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine (Sigma), and 0.5 μg/mL insulin (Sigma)], respectively. For an analysis of the mechanisms involved in Foxc2-mediated regulation of differentiation, PD98059 (Sigma) at 20 μM and 50 μM, and LY294002 (Sigma) at 10 μM and 50 μM were added in the differentiation medium.
A lentiviral vector expressing human Foxc2 and a control vector expressing GFP were purchased from Shanghai GeneChem Co., China. The lentiviruses were propagated in 293T cells and the final titer of virus was 2×108 TU/ml. The multiplicity of infection (MOI) was defined as the ratio of the total number of PFU used in a particular infection to the total number of cells to be transfected.
Third-passage cells were transfected with lentivirus vectors containing either GFP or Foxc2 overnight at MOIs of 10, 50, 100 and 200 plaque-forming units (PFU)/cell. The efficiency of transfection was evaluated under a fluorescence microscope, followed by replacement of the culture medium with fresh L-DMEM supplemented with 10% FBS. Cells were used for the following experiments 72 hours after transfection.
The transfected BMSCs were seeded in 96-well plates at a density of 5× 104 cells/well and cultured for 7 days. MTT assay (Sigma) was performed following the manufacturer's instructions to detect cell proliferation.
Cells were washed with cold PBS (pH 7.0) and lysed at 4°C in lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors]. The samples were heated at 95°C for 5 min and loaded on 12% SDS-PAGE gels and transferred onto methanol-activated PVDF membranes (Millipore, Bedford, MA, USA). After blockage with 5% nonfat milk in TBST (Tris-buffered saline plus 0.1% Tween 20), the membranes were incubated with primary antibodies against Foxc2, OCN, Runx2, VEGF, PDGF-β, ERK, PI3K and β-actin, followed by incubation with the corresponding secondary antibodies. The bands were visualized by using an ECL chemiluminescence kit (Santa Cruz, CA).
Total RNA of cells was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. After reverse transcription reaction, template DNA was used in gene-specific PCR for Foxc2, OCN, Runx2, VEGF and PDGF-β. The primer sequences used for this analysis are listed in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The conditions of real time-PCR were as follows: 40 cycles at 94°C for 5 s and 60°C for 34 s. Dissociation stage was added to the end of amplification procedure. There was no nonspecific amplification determined by the dissolve curve.
BMSCs were fixed and treated with 50 μg/ml 4, 6-diamidino-2-phenyl-indol dihydrochlor-ide (DAPI) for nuclear staining two weeks after transfection. The cells were then stained with OCN, Runx2, VEGF and PDGF-β visualized with a TRITC-conjugated secondary antibody. The primary antibodies were diluted 1:100. Controls included staining without primary antibodies. Fluorescence images were obtained using a fluorescence microcope (fluoview 400, Olympus).
After 2 weeks of transfection, ALP staining was performed using a ALP staining kit (Renbao, Shanghai, China) following the procedures provided by the manufacturer, and ALP activity was determined by the conversion of a colorless p-nitrophenyl phosphate (Pnpp, Sigma, St. Louis, USA ) to a colored p-nitrophenol [22]. For Alizarin red S staining, cells were fixed in 10% formalin and stained with 2% Alizarin red S (Ph 4.0) solution.
Unless otherwise specified, results were presented as mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. P<0.05 was considered statistically significant.
CD44 and CD34 were chosen as markers flow cytometry. BMSCs were successfully expanded 3–4 days after initial seeding, and rapidly expanded into colonies of confluent spindle cells at 10–14 days. The third-passage cells were incubated with antibodies of both CD44 and CD34 (Figure 1A). Results showed that the cells were positive to CD44 (99.56%) and negative to CD34 (0.44%). Matrix mineralization and fat droplet were visualized 10 days after Alizarin red S staining and Oil Red O staining (Figure 1B, C). According to the minimal criteria represented by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy [23], the cultured cells were effectively BMSCs.
Characterization of rat BMSCs. (A) The results of cell surface antigen detection showed that BMSCs were positive to CD44 (99.56%) and negative to CD34 (0.44%). (B) The mineralization nodes were monitored by Alizarin red S staining. (C) Fat droplets formation was monitored by Oil Red O staining.
When BMSCs grew to 80% confluence, the cells were transfected with Lv-GFP for 72 h at various MOIs (10, 50, 100 and 200). The efficiency of gene transfection was determined by examining GFP-positive cells under a fluorescence microscope. The percentages of GFP-positive cells at different MOIs were 27.3±1.5%, 73.4±1.6%, 91.2±1.8% and 90.8±1.5%, respectively (Figure 2A). The MTT assay data indicated that cells overexpressing Foxc2 proliferated significantly (Figure 2B).
Expression of GFP and Foxc2 genes after transfection. (A) Transfection efficiency at various MOIs (10, 50, 100 and 200) 72 hours after transfection (×100). (B) Cell proliferation after transfection in the Lv-Foxc2 and Lv-GFP groups detected using MTT assay. (C) Foxc2 gene expression measured by real-time PCR 72 hours after transfection. GAPDH was used as a housekeeping gene. (D) Foxc2 protein expression evaluated by western blot 72 hours after transfection. Results were expressed as mean±SD of triplicate experiments. #p<0.01 vs. control.
Real time-PCR and Western blot demonstrated that Foxc2 gene was stably expressed in cells 72 h post-transfection (Figure 2C, D). Foxc2 expression was significantly higher in Lv-Foxc2 transfected cells than in Lv-GFP transfected cells (P < 0.05).
Alizarin red S staining showed that the area of positive staining of Lv-Foxc2 transfected cells was nearly 2.5 folds compared with that of the Lv-GFP transfected (Figure 3A, B). ALP expression was evidently observed in Lv-Foxc2 transfected cells after 2 weeks of induction (Figure 3C), and the ALP activity increased to nearly three folds of that in Lv-GFP transfected cells (Figure 3D). Similar expressions of osteoblast differentiation markers, OCN and Runx2, were observed in the two groups of cells (Figure 3E-H). These results demonstrated that Foxc2 overexpression enhanced osteogenesis of BMSCs.
The effect of Foxc2 on osteogenic differentiation. (A, B and C) BMSCs were transfected with Lv-GFP or Lv-Foxc2 for 2 weeks. Cells stained with Alizarin red S (A and B, a1 presents the Lv-Foxc2 group, and a2 presents the Lv-GFP group). Cells stained with ALP (C, c1 presents the Lv-Foxc2 group and c2 presents the Lv-GFP group). (D) ALP activities in BMSCs. (E) Expressions of CON and Runx2 mRNA detected by real-time PCR 72 hours after transfection. The relative density of each gene was statistically analyzed. #p<0.01 vs. control. (F and G) CON and Runx2 expressions examined by Western blot 72 hours after transfection. The results were indicated by the ratio of band intensity of CON or Runx2 with β-actin. (H) Immunostaining for OCN and Runx2 (Texas-Red) with nuclear counterstained (DAPI-blue) 2 weeks after transfection (× 400). The results were expressed as mean±SD of triplicate experiments. #p<0.01 vs. control.
Results of real-time PCR, Western blot and immunostainning showed that the angiogenic markers, VEGF and PDGF-β, were highly expressed in Lv-Foxc2 transfected cells while rarely expressed in Lv-GFP transfected cells (Figure 4). This suggested that Foxc2 hyper-activation might provide the transfected cells a pro-angiogenetic inclination. The VEGF-activated intracellular pathways, PI3K and ERK, can modulate the transcriptional activity of Foxc proteins in Dll4 and Hey2 induction. To investigate the role of the two signaling pathways in the regulation of angiogenesis by Foxc2, the transfected BMSCs were induced to differentiate with or without adding the inhibitors of ERK or PI3K. The results showed that the addition of the specific inhibitor of ERK or PI3K, namely, PD98059 (20 and 50 μM) or LY294002 (10 and 50 μM), led to a decrease in the gene expressions of VEGF and PDGF-β in cells overexpressing Foxc2 (Figure 5A-F). This indicated that inhibition of ERK or PI3K interfered with the regulation of Foxc2 in BMSC differentiation.
Effect of blockade of ERK or PI3K on the expressions of angiogenic makers. Cells overexpressing Foxc2 were cultured in osteogenic induction medium in the absence or presence of PD98059 (A, B, C) or LY294002 (D, E, F). Western blot results of VEGF or PDGF-β expression were indicated by the ratio of band intensity of VEGF or PDGF-β with β-actin (A, B, D, E). The results were expressed as mean±SD of triplicate experiments. Real time-PCR analysis of VEGF or PDGF-β was performed (C, F). The relative mRNA level of each gene was quantitated and statistically analyzed. *p<0.05 vs. control; #p<0.01 vs. control.
The levels of ERK and PI3K were higher in the cells transfected with Lv-Foxc2, indicating that Foxc2 overexpression activated the two signaling pathways in differentiation medium, but the activation was significantly abrogated by pre-treating cells with PD98059 or LY294002 (Figure 6A, B).
Foxc2 overexpression induces activation of ERK and PI3K in BMSCs. (A, B) BMSCs were transfected with Lv-GFP, Lv-Foxc2 or Lv-Foxc2 following pretreatment with 50 μM PD98059 or 10 μM LY294002 for 1 h. Cell lysates harvested after 2 weeks of culture were analyzed by SDS-PAGE and Western blot with anti ERK or anti-PI3K antibody. The result of ERK or PI3K expression was indicated by the ratio of band intensity of ERK or PI3K with β-actin. *p<0.05 vs. Lv-GFP group; #p<0.01 vs. group.
In this study, we demonstrate that Foxc2 overexpression enhances osteogenesis of BMSCs and provides the cells a major pro-angiogenetic inclination. This is consistent with the research of Arnold Caplan, et al. [24]. Our results also show that Foxc2 plays an important regulative role in angiogenesis via activating ERK or PI3K signaling pathway.
The Forkhead protein Foxc2 has emerged as an important regulator of epithelial-to-mesenchymal transitions (EMTs). In the execution of EMT, many genes involved in cell adhesion, mesenchymal differentiation, cell migration, and cell invasion are transcriptionally altered. The functional loss of E-cadherin in an epithelial cell is considered a hallmark of EMT. Many EMT-inducing transcription factors, including Snail, Slug, dEF1, ZEB2, Twist1, Foxc2 and Goosecoid, can repress E-cadherin directly or indirectly when overexpressed [25]. EMT-derived cells share many properties with mesenchymal stem cells (MSCs), while conversely, MSCs express some EMT-associated genes, such as Snail and Foxc2. Similar to MSCs, EMT-derived cells can also differentiate into mature osteoblasts, adipocytes and chondrocytes [26]. Moreover, EMT is an important source for the accumulation of carcinoma associated fibroblasts [27]. It has been proved that the differentiation of epithelial cells is correlated with increased levels of cytoplasmic Foxc2, whereas the dedifferentiation is associated with decreased Foxc2 levels [28]. However, the function of Foxc2 in osteoblast differentiation and angiogenesis has been rarely studied.
We have found in our study that Foxc2 is highly expressed in BMSCs after transfection, and the Foxc2 hyperexpression promotes cell viability (Figure 2). We also reconfirm that Foxc2 enhances the differentiation of BMSCs into osteoblasts (Figure 3). The mechanisms of Foxc2-regulated osteogenesis are still not completely understood. It was reported that the activation of canonical Wnt-β-catenin signals might be involved in the Foxc2-mediated osteoblast differentiation [29]. In addition, Su Jin Park et al. found that Foxc2 was a downstream target of well known anabolic systemic hormones such as BMP2 and PTH, and that Foxc2 promoted osteoblastogenesis by regulating the survival, proliferation and differentiation of osteoblasts through the up-regulation of integrin β1 [30].
Foxc2 also plays an important role in vascular endothelial development. It was reported that Foxc2 acted upstream of Notch signaling in arterial gene expression by directly regulating the Dll4 promoter via a Fox-binding element [31, 32]. Bone marrow-derived cells transfected with Foxc2 caused an increased cellular mobility through the up-regulation of CXCR4 [33]. This increase in cellular mobility due to Foxc2 overexpression has been experienced in other two separate studies, in which the action of Foxc2 was linked to the regulation of two different proteins, p120-catenin and β3 integrin [34, 35]. p120-catenin is a regulator of E-cadherin, which promotes cell-cell adhesion at adherens junctions. Loss of p120-catenin expression results in the destabilization of the E-cadherin complex, which is a critical step in invasion and metastasis [34]. Physiologically, Foxc2 is activated by hypoxia and VEGF. It acts on specific ligand proteins, including p120-catenin and β3 integrin, to destabilize them and promote cellular releasing from their environment. It induces CXCR4 transcription, allowing cells to migrate to the site of injury or hypoxia following a chemokine (CXCL12) gradient. High CXCR4 levels in tumors are linked to poor survival [35].
We have found that Foxc2 overexpression enhances the expressions of angiogenic factors such as VEGF and PDGF-β (Figures 4, 5), and increases the protein levels of ERK and PI3K. The ERK or PI3K inhibitor, PD98059 or LY294002, attenuates the Foxc2-mediated enhancement of angiogenic factors (Figures 5, 6). It has been found that VEGF-activated PI3K and ERK pathways modulate the transcriptional activation of Dll4 and Hey2 genes by Foxc proteins [36]. A recent research shows that VEGF-stimulated PI3K and ERK pathways modulate the transcriptional activity of Foxc2 for arterial gene expression in endothelial cells [37]. Consequently, functional interaction between VEGF signaling and Foxc2 may take place in some aspects of blood vessel formation.
In most in vitro contexts, the PI3K and ERK pathways are stimulated by VEGF together and often act in a synergistic manner. PI3K activation leads to AKT activation, which promotes the migration and survival of endothelial cells and nitric oxide production. ERK/MAPK activation promotes endothelial cell proliferation. Nonetheless, in certain endothelial culture systems, the PI3K branch antagonizes the ERK/MAPK branch [38]. The reasons for the discrepancy of the functional effects of ERK and PI3K are unclear.
MSCs, when are on a stiff substrate and in large numbers, tend to spontaneously differentiate over time into osteoblasts, and this process may be speeded by the overexpression of an important transcriptional factor, Foxc2 [39]. However, many data suggest that the up-regulation of Foxc2, or its transfection, leads to an increase of cellular mobility often linked with progression, invasion and angiogensis of tumor [19, 20, 33]. Thus, the clinical safety of Foxc2-based therapy should still be verified.
Taken together, this work examines the effects of Foxc2 on the commitment of SD rat BMSCs into the osteogenic and angiogenic lineages in vitro. Our results demonstrate that Foxc2 overexpression acts on the transfected BMSCs to enhance the expressions of osteogenic makers and provide the cells a pro-angiogenetic inclination. Furthermore, it is likely that ERK and PI3K signaling pathways are involved in the Foxc2-mediated regulation of angiogenetic inclination.
Future research on the function of Foxc2 in osteogenesis and angiogenesis can be conducted on some novel cell models to study its effects on different mesenchymal-related differentiation processes.
WY and KW are the principal investigators. WY and KW contributed to conception and design, acquisition, analysis and interpretation of data and were involved in drafting the manuscript. WY, HG and DD carried out the experiments. LF and CW were responsible for the statistical analysis and evaluation of the data. All authors read and approved the final manuscript.
Department of Orthopedics, The Second Affilliated Hospital of Xi’an Jiaotong University, Xiwu Road, Xi’an, Shaanxi Province, 710004, China
Department of Orthopedics, The Ninth Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710000, China
Uchida S, Sakai A, Kudo H: Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone. 2003, 32: 491-501. 10.1016/S8756-3282(03)00053-X.View ArticlePubMedGoogle Scholar
Graduate students Hussain Fatakdawala (Marcu Lab), Elena Foster (Revzin Lab) and Priscilla Williams (Silva Lab) won awards for their presentations at the 14th Annual UC Systemwide Bioengineering Symposium.