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1.  In vitro differentiation of bone marrow derived porcine mesenchymal stem cells into endothelial cells 
Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in-vitro. The cultured bone marrow derived cells were CD11b−CD34−CD44+CD45−CD90+ and showed mesodermal lineage differentiation which are characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth media (EGM) with and without high concentrations of VEGF (EGM+VEGF; 50ng/ml). The endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE-cadherin, PECAM-1, CD34, VEGF-R1 and VEGF-R2 after 10days of stimulation. The cells cultured in EGM+VEGF media demonstrated higher amount of DiI-AcLDL positive cells, and enhanced presence of vWF (90%), VE-Cadherin (60%) and PECAM-1 (48%) positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2 fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM+VEGF media when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in-vitro inducing conditions. The differentiated cells would provide new options for re-endothelialization following interventional procedures and tissue engineering.
PMCID: PMC4089894  PMID: 22605545
2.  Vitamin D Supplementation Reduces Airway Hyperresponsiveness and Allergic Airway Inflammation in a Murine Model 
Asthma is a chronic disease associated with airway hyperresponsiveness (AHR), airway obstruction, and airway remodeling. NF-κB is a transcriptional factor that regulates and co-ordinates the expression of various inflammatory genes. The NF-κB subunits, p50 and Rel-A, are translocated to the nucleus by importin α3 and importin α4. There is growing evidence that vitamin D is a potent immunomodulator. However, the evidence for beneficial or adverse effects of vitamin D in asthma is still unclear.
In this study, we examined the effect of vitamin D status on AHR, airway inflammation, and cytokines in the bronchoalveolar lavage fluid (BALF) in a murine model of allergic asthma.
Female BALB/c mice were fed with special vitamin D-deficient or vitamin D-sufficient (2,000 IU/kg) or vitamin D-supplemented (10,000 IU/kg) diet for 13 weeks. Mice were sensitized and challenged with ovalbumin. The effect of vitamin D on lung histology, AHR, T-regulatory cells and BALF cytokines was examined. The expression of importin-α3 and Rel-A in the lung of OVA-sensitized mice was analyzed using immunofluorescence.
Vitamin D deficiency was associated with higher AHR in OVA-sensitized and challenged mice than those in vitamin D-sufficient mice. This was accompanied with marked signs of airway remodeling, high BALF eosinophilia, increased BALF pro-inflammatory cytokines, reduced BALF IL-10 levels, reduced blood T-regulatory cells, increased expression of importin-α3 and Rel-A in the lung tissue. Vitamin D supplementation attenuated the pro-inflammatory effects, but did not completely reverse the features of allergic airway inflammation.
Conclusion and Clinical Relevance
Vitamin D could be beneficial as an adjunct therapy in the treatment of allergic asthma.
PMCID: PMC3671499  PMID: 23711130
3.  Vitamin D and the Epidemiology of Upper Gastrointestinal Cancers: A Critical Analysis of the Current Evidence 
Prospective analyses have yet to uncover a consistent relationship between vitamin D status and incidence and mortality of rarer cancers including esophageal and upper gastrointestinal cancers. We searched PubMed for literature regarding the epidemiology of upper gastrointestinal cancers and vitamin D published over the last decade and then summarized and critiqued the results of these studies in this review. The search yielded nine relevant studies. Overall, no consistent relationship was reported between serum vitamin D levels or a surrogate and upper gastrointestinal cancers. Four studies reported negative correlations between vitamin D status and upper gastrointestinal cancer, three reported positive correlations, one reported no correlation, and one reported both positive and negative correlations. No relationship has been established based on epidemiological data, but studies examining sun exposure consistently report an inverse association with esophageal cancer. The current literature is limited by the methods used to assess vitamin D status, lack of specific data for the types of upper gastrointestinal cancer, and failure to establish a temporal relationship between vitamin D status assessment and presentation of upper gastrointestinal cancer. It is possible that the lack of a consistent relationship is a consequence of inaccurate and imprecise assessment of vitamin D status.
PMCID: PMC3681828  PMID: 23563888
Cancer; Epidemiology; Esophagus; Gastrointestinal; Vitamin D
4.  Vitamin D and inflammatory diseases 
Beyond its critical function in calcium homeostasis, vitamin D has recently been found to play an important role in the modulation of the immune/inflammation system via regulating the production of inflammatory cytokines and inhibiting the proliferation of proinflammatory cells, both of which are crucial for the pathogenesis of inflammatory diseases. Several studies have associated lower vitamin D status with increased risk and unfavorable outcome of acute infections. Vitamin D supplementation bolsters clinical responses to acute infection. Moreover, chronic inflammatory diseases, such as atherosclerosis-related cardiovascular disease, asthma, inflammatory bowel disease, chronic kidney disease, nonalcoholic fatty liver disease, and others, tend to have lower vitamin D status, which may play a pleiotropic role in the pathogenesis of the diseases. In this article, we review recent epidemiological and interventional studies of vitamin D in various inflammatory diseases. The potential mechanisms of vitamin D in regulating immune/inflammatory responses in inflammatory diseases are also discussed.
PMCID: PMC4070857  PMID: 24971027
asthma; atherosclerosis; chronic kidney disease; inflammatory bowel disease
5.  Importins and Exportins Regulating Allergic Immune Responses 
Mediators of Inflammation  2014;2014:476357.
Nucleocytoplasmic shuttling of macromolecules is a well-controlled process involving importins and exportins. These karyopherins recognize and bind to receptor-mediated intracellular signals through specific signal sequences that are present on cargo proteins and transport into and out of the nucleus through nuclear pore complexes. Nuclear localization signals (NLS) present on cargo molecules to be imported while nuclear export signals (NES) on the molecules to be exported are recognized by importins and exportins, respectively. The classical NLS are found on many transcription factors and molecules that are involved in the pathogenesis of allergic diseases. In addition, several immune modulators, including corticosteroids and vitamin D, elicit their cellular responses by regulating the expression and activity of importin molecules. In this review article, we provide a comprehensive list of importin and exportin molecules and their specific cargo that shuttled between cytoplasm and the nucleus. We also critically review the role and regulation of specific importin and exportin involved in the transport of activated transcription factors in allergic diseases, the underlying molecular mechanisms, and the potential target sites for developing better therapeutic approaches.
PMCID: PMC3964845  PMID: 24733961
6.  MMP‐1 and MMP‐9 regulate epidermal growth factor‐dependent collagen loss in human carotid plaque smooth muscle cells 
Physiological Reports  2014;2(2):e00224.
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.
This report described the underlying mechanisms by which MMP‐1 and MMP‐9 induced by EFGR activation decreases the interstitial collagens and this could result in plaque instability in patients with carotid stenosis. Thus, selective blockade of EGFR and/or MMP‐9 may be a novel strategy and a promising target to stabilize atherosclerotic plaques and thus decreases morbidity and mortality.
PMCID: PMC3966234  PMID: 24744893
Atherosclerosis; carotid plaques; interstitial collagens; matrix metalloproteinase; vascular smooth muscle cells
7.  NPY and NPY Receptors in Airway Structural and Inflammatory Cells in Allergic Asthma 
Neuropeptide Y (NPY) level is elevated in allergic asthmatic airways and activation of NPY receptor-1 (NPY-Y1) on antigen presenting cells (APCs) is essential for T-cell priming. Paradoxically, NPY-Y1 modulates hyper-responsiveness in T cells, suggesting a bimodal role for NPY in APCs and T-cells. Therefore, determination of the temporal and spatial expression pattern of NPY and its receptors in asthmatic airways is essential to further understand the role of NPY in allergic asthma.
Lungs were isolated from control and acute and chronic stages of OVA-sensitized and challenged mice (OVA). Stains, including H&E, PAS, and trichrome, were used to determine the severity of lung pathology. The expression patterns of NPY and NPY-Y receptors in the airways were determined using ELISA and immunofluorescence. Cytokine levels in the BALF were also measured.
NPY levels were undetectable in the BALF of control mice, but significantly increased in the OVA group at day 80. Levels of IL-4, TGF-β1 and TGF-β2, significantly increased and peaked on day 45 and decreased on day 80 in the OVA group, exhibiting an inverse correlation with NPY levels. NPY expression was localized to macrophage-like cells in the peri-bronchial and peri-vascular areas in the lung tissue. NPY-Y1 and -Y5 receptors were constitutively expressed by both structural and inflammatory cells in the lung tissue.
NPY produced by activated macrophage-like cells may be involved in regulating cytokine production and cellular activities of immune cells in asthma. However, it remains unclear whether such an increase in NPY is a defensive/compensatory mechanism to modulate the effects of inflammatory cytokines.
PMCID: PMC3488603  PMID: 22705097
Allergic asthma; antigen presenting cells (APCs); macrophage; neuropeptide Y (NPY); NPY receptor-Y1; transforming growth factor beta (TGF-β)
8.  Vitamin D Receptor Expression in the Mucosal Tissue at the Gastroesophageal Junction 
Barrett’s Esophagus is considered to be a precursor to adenocarcinoma and the information on VDR expression in normal and Barrett’s esophagus is significantly lacking. In this study, we examined the expression of VDR in the lower esophagus and gastric cardia of normal and Barrett’s esophagus by immunofluorescence. Columnar mucosa but not squamous mucosa at the gastroesophageal junction showed positive immunofluorescence to VDR. Submucosal glands and ducts deep to the normal squamous mucosa stained positive for VDR and localized in the cytoplasm and perinuclear regions with no nuclear staining. Interestingly, the Barrett’s mucosa stained strongly positive for VDR. Glandular structures in the mucosal layer were far less abundant in the Barrett’s mucosa than in the normal gastric mucosa. As a result, fewer structures deep to the Barrett’s epithelial layer stained positive for VDR when compared to normal gastric mucosa. These findings suggest that in normal esophagus VDR expression is restricted to columnar epithelium and glandular structures. Furthermore, strong VDR expression in Barrett’s mucosa may indicate an increased sensitivity of this tissue to endogenous or therapeutic effects of Vitamin D.
PMCID: PMC3411275  PMID: 22664272
Barrett’s esophagus; Epithelium; Gastroesophageal junction; Vitamin D; Vitamin D receptor
9.  Calcitriol Decreases Expression of Importin α3 and Attenuates RelA Translocation in Human Bronchial Smooth Muscle Cells 
Journal of clinical immunology  2012;32(5):1093-1103.
A potent immunomodulatory role of Vitamin D in both innate and adaptive immunity has recently been appreciated. In allergic asthma, activation of NF-κB induces transcription of various cytokines and chemokines involved in allergic airway inflammation. The nuclear import of activated NF-κB p50/RelA subunit is dependent on importin α3 (KPNA4) and importin α4 (KPNA3). In this study, we examined the role of importin α3 in immunomodulatory effect of calcitriol in human bronchial smooth muscle cells (HBSMCs).
Cultured HBSMCs were stimulated with calcitriol in the presence and absence of cytokines, TNF-α, IL-1β, and IL-10. The mRNA transcripts of importin α3 and α4 were analyzed using qPCR while protein expression of importin α3, α4 and nuclear RelA was analyzed by immunoblotting.
Calcitriol significantly decreased mRNA and protein expression of importin α3 as well as nuclear protein expression of NF-κB p65 (RelA). The decreased activation of RelA by calcitriol was confirmed by decreased release of RelA-inducible molecules, including IL-5, IL-6 and IL-8, by HBSMCs upon calcitriol treatment. Calcitriol attenuated the effect of TNF-α and IL-1β to upregulate mRNA and protein expression of importin α3. IL-10 significantly decreased the TNF-α induced expression of importin α3 and this effect was further potentiated by calcitriol.
These data suggest that under inflammatory conditions, calcitriol decreases the expression of importin α3 resulting in decreased nuclear import of activated RelA. This could be a novel mechanism by which calcitriol could exert its immunomodulatory effects to reduce allergic airway inflammation and thus may alleviate the symptoms in allergic asthma.
PMCID: PMC3444658  PMID: 22526597
Active vitamin D; Allergic airway inflammation; Asthma; Bronchial smooth muscle cells; Calcitriol; Importinα3 (KPNA4); Importinα4 (KPNA3); NF-κB; Vitamin D receptor (VDR)
10.  Vitamin D Receptor Expression and Neoadjuvant Therapy in Esophageal Adenocarcinoma 
Esophageal adenocarcinoma carries a poor prognosis. Tumor response to neoadjuvant therapy is a key prognostic factor in patients with adenocarcinoma of the esophagus, but is inconsistent. Identifying tumor characteristics that portend a favorable response to neoadjuvant therapy would be a valuable clinical tool. The anticancer actions of vitamin D and its receptor may have implications. In this study, 15 biopsy specimens were procured retrospectively from patients being treated for adenocarcinoma of the esophagus. The tissue was immunostained for the vitamin D receptor and compared on the basis of response to neoadjuvant therapy. Tumors that did not respond to neoadjuvant therapy had greater expression of VDR than tumors that responded completely. Expression of VDR declined with tumor de-differentiation. The data suggest that a relationship between vitamin D receptor expression and response to neoadjuvant therapy is plausible.
PMCID: PMC3400680  PMID: 22546272
Adenocarcinoma; Esophageal cancer; Neoadjuvant therapy; Vitamin D; Vitamin D receptor
11.  Vitamin D deficiency decreases the expression of VDR and prohibitin in the lungs of mice with allergic airway inflammation 
Asthma is one of the most common chronic inflammatory diseases of the airways. Calcitriol exerts its action through Vitamin D receptor (VDR), which is a high affinity nuclear receptor. VDR is a transcription factor that alters the transcription of target genes which are involved in a wide spectrum of biological responses. Lower serum vitamin D levels are associated with airway hyperresponsiveness and increased asthma severity. Prohibitin is a ubiquitously expressed protein localized to the cell and mitochondrial membranes and the nucleus.
Methods and results
HBSMCs were cultured and treated with calcitriol and/or TNF-α. The mRNA and protein expression of prohibitin and VDR were analyzed using qPCR and immunoblotting, respectively. In the in vivo studies, female BALB/c mice were fed with special vitamin D-deficient or 2,000 IU/kg of vitamin D-supplemented diet for 13 weeks. Mouse model of allergic airway inflammation was developed by OVA-sensitization and challenge. The expression pattern of TNF-α, prohibitin and VDR in the lung of OVA-sensitized mice was analyzed using immunofluorescence. Calcitriol significantly increased and TNF-α decreased the protein and mRNA expression of prohibitin and VDR in HBSMCs. There was significantly increased expression of TNF-α and decreased expression of VDR and prohibitin in the lung of vitamin D-deficient mouse model of allergic airway inflammation.
These results suggest that under inflammatory conditions there is decreased expression of VDR resulting in decreased expression of prohibitin, which is a vitamin D target gene. Vitamin D deficiency causes increase in the expression of TNF-α, thereby increasing inflammation and decreases the expression of VDR and prohibitin. Supplementation with vitamin D reduces the levels of TNF-α, thereby increasing the expression of VDR and prohibitin that could be responsible for reducing allergic airway inflammation.
PMCID: PMC3410542  PMID: 22537547
Allergic airway inflammation; Asthma; Human Bronchial Smooth Cells; Inflammation; Prohibitin; TNF-α; Vitamin D receptor; Vitamin D
12.  Vitamin D Deficiency Induces Cardiac Hypertrophy and Inflammation in Epicardial Adipose Tissue in Hypercholesterolemic Swine 
Vitamin D is a sectosteroid that functions through Vitamin D receptor (VDR), a transcription factor, which controls the transcription of many targets genes. Vitamin D deficiency has been linked with cardiovascular diseases, including heart failure and coronary artery disease. Suppressor of cytokine signaling (SOCS)3 regulates different biological processes such as inflammation and cellular differentiation and is an endogenous negative regulator of cardiac hypertrophy.
The purpose of this study was to test the hypothesis that vitamin D deficiency causes cardiomyocyte hypertrophy and increased proinflammatory profile in epicardial adipose tissue(EAT), and this correlates with decreased expression of SOCS3 in cardiomyocytes and EAT.
Eight female Yucatan miniswine were fed vitamin D-sufficient (900 IU/d) or vitamin D-deficient hypercholesterolemic diet. Lipid profile, metabolic panel, and serum 25(OH)D levels were regularly measured. After 12 months animals were euthanized and histological, immunohistochemical and qPCR studies were performed on myocardium and epicardial fat.
Histological studies showed cardiac hypertrophy, as judged by cardiac myocyte cross sectional area, in vitamin D-deficient group. Immunohistochemical and qPCR analyses showed significantly decreased mRNA and protein expression of VDR and SOCS3 in cardiomyocytes of vitamin D-deficient animals. EAT from vitamin D-deficient group had significantly higher expression of TNF-α, IL-6, MCP-1, and decreased adiponectin in association with increased inflammatory cellular infiltrate. Interestingly, EAT from vitamin D-deficient group had significantly decreased expression of SOCS3.
These data suggest that vitamin D deficiency induces hypertrophy in cardiomyocytes which is associated with decreased expression of VDR and SOCS3. Vitamin D deficiency is also associated with increased inflammatory markers in EAT. Activity of VDR in the body is controlled through regulation of vitamin D metabolites. Therefore, restoration of VDR function by supplementation of VDR ligands in vitamin D- deficient population might be helpful in reducing inflammation and cardiovascular risk.
PMCID: PMC3411274  PMID: 22537546
Cardiac Hypertrophy; Coronary Artery Disease; Epicardial Fat; Inflammation; Vitamin D
13.  Successful Transfection of Genes Using AAV-2/9 Vector in Swine Coronary and Peripheral Arteries 
The Journal of Surgical Research  2011;175(1):169-175.
Gene therapy has attracted attention for its potential to treat several cardiovascular diseases. The use of adeno-associated viral (AAV) vectors to facilitate therapeutic gene transfer to suppress intimal hyperplasia is a promising concept. The objective of this study was to analyze the in vivo transduction of a novel recombinant AAV-2/9 vector with SM22α promoter, containing β-galactosidase gene (Lac Z) or green fluorescent protein (GFP) as reporter genes, to the medial layer smooth muscle cells (SMCs) of swine coronary and peripheral arteries.
The AAV2/9 vector containing SM22α (1×1013 pfu) were administered into carotid/femoral/coronary arteries of domestic swine using irrigating balloon catheter-based gene delivery. Following gene transfer, cryosections of arteries were processed for X-Gal and GFP analysis. Fluorescence microscopy and Western blotting were done to analyze the GFP expression in the SMCs.
LacZ mRNA expression was visualized in the medial layer 7 days after vector administration. The GFP expression was detected at 7th day and lasted for at least 2 months showing the longer-lasting expression of the AAV2/9-vector. Control arteries did not show any expression of GFP or LacZ. There was no significant effect of AAV2/9 viral transduction on serum amylase, fibrinogen and serum CRP levels.
These finding support the use of AAV2/9 as a vector to effectively transduce a gene in SMCs of coronary and peripheral arteries without causing inflammation.
PMCID: PMC3150285  PMID: 21529824
Adeno-associated virus; Gene therapy; Intimal hyperplasia; Restenosis; Vascular smooth muscle cells; Vector
14.  Hematopoietic Stem and Progenitor Cells in Inflammation and Allergy 
Hematopoietic stem and progenitor cells contribute to allergic inflammation. Pro-inflammatory cytokines that are generated following allergen challenge can impact the differentiation of hematopoietic progenitor cells leading to increased production of effector cells such as eosinophils and basophils, which are key cells involved in the pathogenesis of allergic airway inflammation. Homing of stem cells to the lungs is associated with inflammatory and remodeling changes in asthmatics. Factors that modulate the differentiation and increased migration of stem cells to the site of inflammation in asthma remain to be defined. Stem cells can mature at the site of inflammation in response to inflammatory mediators and other components in the milieu. While the available data suggest that hematopoietic cells traffic to target tissues, the molecular factors underlying in situ differentiation have yet to be specified. Here, we critically evaluate the potential role of hematopoietic progenitors in contributing to the increased immune cell infiltrate in allergic asthma and the factors that drive their differentiation.
PMCID: PMC3849597  PMID: 24363657
allergic asthma; hematopoietic stem/progenitor cells; inflammation; eosinophils; fibrocytes
15.  Calcium-Activated Potassium Channel KCa3.1 in Lung Dendritic Cell Migration 
Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell–mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca2+ and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11chighCD11blow than CD11clowCD11bhigh DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non–Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non–Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO–induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.
PMCID: PMC3262686  PMID: 21493782
allergic airway inflammation; antigen uptake; asthma; calcium-activated potassium channel; dendritic cell
16.  Epidermal Growth Factor Increases LRF/Pokemon Expression in Human Prostate Cancer Cells 
Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis.
PMCID: PMC3175504  PMID: 21640721
prostate cancer; Pokemon; EGF; LRF; ZBTB7
17.  Cross-talk between Angiotensin II and IGF-1-induced Connexin 43 Expression in Human Saphenous Vein Smooth Muscle Cells 
Vascular restenosis following coronary artery bypass graft can cause major clinical complications due to intimal hyperplasia in venous conduits. However, the precise underlying mechanisms of intimal hyperplasia are still unclear. We have recently reported that increased expression of connexin43 (Cx43) is involved in the proliferation of vascular smooth muscle cells (SMCs) in human saphenous vein (SV). In this study, we investigated the signaling transduction pathway involved in Cx43 expression and SV SMC proliferation. Angiotensin-II (AT-II, 100ng/ml) increased angiotensin II receptor 1 (AT-1R) protein expression and insulin-like growth factor-1 (IGF-1) (100ng/ml) upregulated IGF-1 receptor (IGF-1R) protein expression in SV SMCs. Interestingly, AT-1R expression was also increased by IGF-1 treatment, and IGF-1R expression was increased by AT-II treatment, which was blocked by siRNA-IGF-1R and siRNA-AT-1R respectively. Furthermore, the effect of AT-II and IGF-1 signal cross-talk inducing upregulation of their reciprocal receptors was blocked by siRNA against extracellular signal–regulated kinases 1/2 (Erk 1/2) in SMCs of saphenous vein. Moreover, AT-II and IGF-1 induced Cx43 expression via phosphorylation of Erk 1/2 and activation of transcription factor activator protein 1 (AP-1) through their reciprocal receptors in SV SMCs. These data demonstrate a cross-talk between IGF-1R and AT-1R in AT-II and IGF-1-induced Cx43 expression in SV SMCs involving Erk 1/2 and downstream activation of the AP-1 transcription factor.
PMCID: PMC3000445  PMID: 20731749
Angiotensin II; Connexins; Intimal Hyperplasia; Restenosis; Vascular Smooth Muscle Cells; Vein Graft Disease
18.  Suppressor of Cytokine Signaling-3 and Intimal Hyperplasia in Porcine Coronary Arteries following Coronary Intervention 
The growth and differentiation of cells is regulated by cytokines by binding to cell-surface receptors and activating intracellular signal transduction cascade. Suppressor of cytokine signaling (SOCS)-3 is a negative regulator of cytokines. In this study we examined the expression of SOCS3 in porcine coronary artery smooth muscle cells (PCASMCs) in vitro and in proliferating smooth muscle cells of neointimal lesions after coronary artery intervention in a swine model.
Methods and Results
PCASMCs were cultured and stimulated with TNF-α and/or IGF-1 individually or in combination. Protein expression of SOCS-3 was examined using Western blot. For in vivo studies, six female Yucatan miniswine were fed with special high cholesterol diet for 8 months. At 4 months of high cholesterol diet, animals underwent coronary balloon angioplasty. At the end of 8 months animals were euthanized, coronary arteries were isolated and morphological and histological studies were performed. Western blot data revealed significantly high SOCS-3 expression in PCASMCs in the presence of either TNF-α or IGF-1 (5–6 fold) alone. However, in the presence of both TNF-α and IGF-1 the SOCS-3 expression was significantly decreased (4–5 fold). Results from morphological studies including, H&E and Masson’s trichrome stain showed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased proliferating cell nuclear antigen (PCNA) in neointimal lesion. Interestingly, there was significantly decreased expression of SOCS3 in smooth muscle cells of neointima as compared to control.
These data suggest that SOCS3 expression is decreased in proliferating smooth muscle cells of neointimal lesions. This leads to uncontrolled growth of vascular smooth muscle cells in injured arteries leading to restenosis. Therefore, local delivery of SOCS3 gene at the site of injury after coronary artery intervention could regulate the proliferation of vascular smooth muscle cells and help in preventing the neointimal hyperplasia and restenosis.
PMCID: PMC3139760  PMID: 21540027
Angioplasty; Coronary Intervention; Inflammation; Intimal Hyperplasia; Restenosis; Suppressor of cytokine signaling-3
19.  Neuropeptide Y Receptors in Carotid Plaques of Symptomatic and Asymptomatic Patients: Effect of Inflammatory Cytokines 
Cytokines released by the immune cells at the site of plaque milieu induce smooth muscle cell apoptosis to promote plaque instability. But, neuropeptide Y (NPY), a pleotropic factor, may modulate the effects of cytokines in atherosclerotic plaques of patients with carotid stenosis. Our aim was to investigate the relative expression of NPY-Y1, NPY-Y2 and NPY-Y5 receptors on carotid plaque vascular smooth muscle cells (pVSMCs) of symptomatic (S) and asymptomatic (AS) patients and examine the effect of inflammatory cytokines on the expression of NPY receptors, that may attenuate plaque rupture.
Methods and Results
In healthy carotid artery, there was significantly increased immunopositivity and increased mRNA transcripts of NPY-Y1 and NPY-Y5 receptors in thin sections and isolated VSMCs, respectively, compared to S and AS plaques. However, the NPY-Y2 expression was higher in S and AS pVSMCs than controls. Stimulation of the cells with TNF-α, IL-12 or IFN-γ (50 ng/ml) decreased mRNA transcripts of NPY-Y1 and NPY-Y5 and increased NPY-Y2 mRNAs in VSMCs of healthy carotid artery. The effect of the cytokines on mRNA transcripts of NPY-Y5 and NPY-Y2 in pVSMCs of S and AS patients was similar to healthy VSMCs, but with variable effect on NPY-Y1.
Increased expression of NPY-Y2 receptors in symptomatic pVSMCs than in healthy and asymptomatic subjects suggests a potential role of NPY-Y2 in plaque instability. This is further supported by the pronounced effect of atheroma-associated cytokines to increase NPY-Y2 mRNA transcripts in pVSMCs of patients with carotid stenosis.
PMCID: PMC3091977  PMID: 21352822
Atheromatous cytokines; Atherosclerosis; Carotid plaque; Carotid stenosis; Neuropeptide Y; Smooth muscle cells
20.  Increased Expression of Angiopoietins and Tie2 in the Lungs of Chronic Asthmatic Mice 
Angiopoietin (Ang)1 and Ang2 are ligands for Tie2 tyrosine kinase receptor (Tie2). Elevated levels of Ang1 and Ang2 in induced sputum of patients with asthma have been reported, with a positive correlation of Ang2 levels with the severity of airway occlusion. Although studies have shown Tie2-mediated regulation of nonvascular cells in some pathological conditions, current knowledge on Tie2 signaling in asthma is limited to the vasculature. We examined the expression pattern of Ang1, Ang2, vascular endothelial growth factor (VEGF), and Tie2 and their correlation with the degree of airway remodeling in the lung of ovalbumin (OVA)-sensitized and OVA-challenged mice with airway hyperresponsiveness. Lung tissues were isolated from Balb/c mice after OVA sensitization and challenge. Hematoxylin and eosin, periodic acid-Schiff, and trichrome staining were used to show the lung pathology. The expression of Ang1, Ang2, VEGF, and Tie2 was examined using immunofluorescence, Western blot, ELISA, and real-time PCR. In the lung of normal mice, Tie2 expression was detected only in the blood vessels. However, in the lung of OVA-sensitized and OVA-challenged mice, Tie2 was abundantly expressed in airway epithelial cells and in a subset of macrophages in addition to constitutive expression in pulmonary vessels. The increase in Tie2 expression correlated with the severity of airway remodeling. Macrophages and airway epithelial cells express Ang2 and VEGF only in allergic models. Ang1 was constitutively expressed, with a decrease in mRNA level in allergic models. In conclusion, increased expression of Tie2 and Ang2 in allergic airway epithelium and alveolar macrophages correlates with the severity of airway remodeling.
PMCID: PMC3095938  PMID: 20463289
airway epithelial cells; airway remodeling; angiopoietins; Tie2 tyrosine kinase receptor; vascular endothelial growth factor
21.  Tumor Necrosis Factor-α Regulates p27kip Expression and Apoptosis in Smooth Muscle Cells of Human Carotid Plaques via Forkhead Transcription Factor O1 
Apoptosis of vascular smooth muscle cells (SMCs) is controlled by a balance between the effect of growth factors and cytokines, and is involved in plaque instability in advanced atherosclerotic lesions. Recently, we reported high levels of atheroma-associated cytokines, including tumor necrosis factor-α (TNF-α), in carotid plaques of symptomatic patients. These cytokines induce apoptosis of vascular SMCs, and thus could be responsible for plaque rupture, a clinically devastating event. In this study, we examined the effect of TNF-α on the cell cycle inhibitor p27kip and apoptosis of SMCs in human carotid plaques, and the underlying mechanism. Both Forkhead box subclass o1 (FoxO1) and p27kip were more strongly expressed in symptomatic than asymptomatic atherosclerotic plaques. TNF-α significantly induced the expression of FoxO1 in asymptomatic plaque SMCs in a dose- and time-dependent manner via JNK signaling pathway. TNF-α also induced phosphorylation of FoxO1, resulting in its cytoplasmic translocation/nuclear exclusion of transcription factors. The effect of TNF-α was blocked by the PI3K inhibitor, LY294002. Meanwhile, TNF-α not only induced the p27kip expression and cell cycle arrest in G0–G1 phase, but also enhanced caspase-3 activity and induced apoptosis in SMCs of asymptomatic plaques. However, the potential effect of TNF-α on the cell cycle inhibitor p27kip and apoptosis of SMCs was inhibited by siRNA against FoxO1 in asymptomatic patients. These data suggest the involvement of FoxO1 transcription factor in TNF-α-induced expression of a cell cycle regulatory protein and apoptosis of SMCs, thus regulating the stability of atherosclerotic plaques with carotid stenosis.
PMCID: PMC3031176  PMID: 21075101
Apoptosis; Atherosclerosis; Forkhead Transcription Factor; Plaque Stability; Vascular Smooth Muscle Cell
22.  Fms-Like Tyrosine Kinase 3 Ligand Decreases T Helper Type 17 Cells and Suppressors of Cytokine Signaling Proteins in the Lung of House Dust Mite–Sensitized and –Challenged Mice 
We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells and CD4+CD25+ICOS+Foxp3+IL-10+ T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)–sensitized and –challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 μg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)–β in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-β levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4+IL-17+IL-23R+CD25− T cells with high expression of retinoic acid–related orphan receptor (ROR)–γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4+IL-17+IL-23R+CD25− T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-β levels in the BALF, which, in turn, decrease CD4+IL-17+IL-23R+ROR-γt+CD25− T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice.
PMCID: PMC2970852  PMID: 19933379
airway hyperresponsiveness; house dust mite; retinoic acid–related orphan receptor–γt; suppressors of cytokine signaling; T helper cell type 17
23.  Expression of Leukemia/Lymphoma-Related Factor (LRF/POKEMON) in Human Breast Carcinoma and Other Cancers 
The POK family of proteins plays an important role in not only embryonic development and cell differentiation, but also in oncogenesis. Leukemia/lymphoma-related factor (LRF) belongs to the POK family of transcriptional repressors and is also known as POK erythroid myeloid ontogenic factor (POKEMON), which binds to short transcripts of HIV-1 (FBI-1) and TTF-1 interacting peptide (TIP21). Its oncogenic role is known only in lymphoma, non-small cell lung carcinoma, and malignant gliomas. The functional expression of LRF in human breast carcinoma has not yet been confirmed. The aim of this study was to investigate and compare the expression of LRF in human breast cancer tissues and other human tumors. The expression of LRF mRNA transcripts and protein was observed in twenty human benign and malignant breast biopsy tissues. Expression of LRF was observed in several formalin-fixed tissues by immunohistochemistry and immunofluorescence. All malignant breast tissues expressed mRNA transcripts and protein for LRF. However, 40% and 15% benign breast biopsy tissues expressed LRF mRNA transcripts and protein, respectively. The overall expression of LRF mRNA transcripts and total protein was significantly more in malignant breast tissues than the benign breast tissues. LRF expression was also observed in the nuclei of human colon, renal, lung, hepatocellular carcinomas and thymoma tumor cells. In general, a significantly higher expression of LRF was seen in malignant tissues than in the corresponding benign or normal tissue. Further studies are warranted to determine the malignant role of LRF in human breast carcinoma.
PMCID: PMC2939325  PMID: 20471975
Breast Cancer; Pokemon; LRF; ZBTB7; POK
24.  Programmed Death-1 Antibody Blocks Therapeutic Effects of T-Regulatory Cells in Cockroach Antigen-Induced Allergic Asthma 
We recently reported that the adoptive transfer of T-regulatory cells (Tregs) isolated from lung and spleen tissue of green fluorescent protein–transgenic mice reversed airway hyperresponsiveness and airway inflammation. Because Programmed Death-1 (PD-1) is a pivotal receptor regulating effector T-cell activation by Tregs, we evaluated whether PD-1 is involved in the therapeutic effect of naturally occurring Tregs (NTregs) and inducible Tregs (iTregs) in cockroach (CRA)-sensitized and challenged mice. The CD4+CD25+ NTregs and CD4+CD25− iTregs isolated from the lungs and spleens of BALB/c mice were adoptively transferred into CRA-sensitized and CRA-challenged mice with and without anti–PD-1 antibody (100 μg/mice). The CD4+CD25+ T cells in the lung were phenotyped after adoptive transfer. Concentrations of IL-4, IL-5, IL-10, IFN-γ, and IL-13 in bronchoalveolar lavage fluid (BALF) were measured using ELISA. The NTregs and iTregs from either lung or spleen tissue reversed airway hyperresponsiveness for at least 4 wk. However, the therapeutic effect was blocked by administering the anti–PD-1 antibody. The administration of Tregs-recipient mice with anti–PD-1 antibody significantly decreased cytotoxic T-lymphocyte antigen-4 expression, with low concentrations of Forkhead-winged transcriptional factor box 3 (Foxp3) mRNA transcripts in lung CD4+CD25+ T cells. These mice had substantially higher concentrations of BALF IL-4, IL-5, and IL-13, but significantly decreased levels of BALF IL-10. Adoptive therapy recipients without the anti–PD-1 antibody exhibited high levels of CTLA-4 expression and Foxp3 transcripts in lung CD4+CD25+ T cells, with a significant decrease in BALF IL-4, IL-5, and IL-13 concentrations and a substantial increase in BALF IL-10 concentrations. These data suggest that the reversal of airway hyperresponsiveness and airway inflammation by Tregs is mediated in part by PD-1, because other costimulatory molecules (e.g., inducible costimulatory molecule [ICOS] or CTLA-4) have been shown to play a role in Treg-mediated suppression.
PMCID: PMC2951873  PMID: 19901343
airway hyperresponsiveness; airway inflammation; anti–PD-1 antibody; cockroach antigen; Forkhead-winged transcriptional factor box P3
25.  Insulin-like Growth factor-1 Induces Phosphorylation of PI3K-Akt/PKB to Potentiate Proliferation of Smooth Muscle Cells in Human Saphenous Vein 
Coronary revascularization by coronary artery bypass grafting (CABG) is recommended in patients with recurrent myocardial ischemia. However, the long-term results of CABG using saphenous vein (SV) graft, compared to internal mammary artery (IMA) graft, have not been satisfactory. The SV graft failure is due to development of intimal hyperplasia, a process characterized by abnormal migration and proliferation of smooth muscle cells (SMCs) in the intimal layer of the vein graft. Insulin growth factor 1 (IGF-1) is a major mitogenic growth factor released at the site of the shear stress-induced graft injury. This study, for the first time, compares the extent of IGF-1-PI3K-Akt activation in isolated human by pass graft conduits. Human SV and IMA vessels were collected and SMCs isolated and cultured. In cultured SMCs, effect of IGF-1 was examined on total and phosphorylated PI3K, Akt and IGF-1R by Western blot analysis. Cell proliferation was measured using BrdU ELISA. There was no significant difference in the basal expression of phosphorylated PI3K, Akt and IGF-1R in SV and IMA SMCs from human bypass conduits. However, we observed an upregulation of IGF-1 receptors in the SV SMCs in response to IGF-1 stimulation with no effect in IMA SMCs. Furthermore, the immunoblotting and cellular activation of signaling ELISA (CASE) assay demonstrated a significantly higher activity of both PI3K and Akt in IGF-1-stimulated SV SMCs than IMA. This was inhibited by an IGF-1R blocking antibody. IGF-1 induced proliferation in both SV and IMA SMCs was inhibited by a PI3K inhibitor, wortmannin. These data demonstrate differential activity of IGF-1-induced PI3K-Akt activation, which was quantitatively and temporally greater in SV SMCs than in the IMA. This, at least in part, could explain the greater propensity of the SV conduits than the IMA to undergo intimal hyperplasia following CABG.
PMCID: PMC2900498  PMID: 20471974
Coronary artery bypass graft; IGF-1; Internal mammary artery; Intimal hyperplasia; PI3K; Akt/PKB; Proliferation; Restenosis; Saphenous vein; Vascular smooth muscle cells

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