Abstract Objectives Delayed paraplegia secondary to ischemia–reperfusion injury is a devastating complication of thoracoabdominal aortic surgery. Alpha-2 agonists have been shown to attenuate ...ischemia–reperfusion injury, but the mechanism for protection has yet to be elucidated. A growing body of evidence suggests that astrocytes play a critical role in neuroprotection by release of neurotrophins. We hypothesize that alpha-2 agonism with dexmedetomidine increases glial cell-line–derived neurotrophic factor in spinal cord astrocytes to provide spinal cord protection. Methods Spinal cords were isolated en bloc from C57BL/6 mice, and primary spinal cord astrocytes and neurons were selected for and grown separately in culture. Astrocytes were treated with dexmedetomidine, and glial cell-line–derived neurotrophic factor was tested for by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess neuronal viability. Results Spinal cord primary astrocytes treated with dexmedetomidine at 1 μmol/L and 10 μmol/L had significantly increased glial cell-line–derived neurotrophic factor production compared with control ( P < .05). Neurons subjected to oxygen glucose deprivation had significant preservation ( P < .05) of viability with use of dexmedetomidine-treated astrocyte media. Glial cell-line–derived neurotrophic factor neutralizing antibody eliminated the protective effects of the dexmedetomidine-treated astrocyte media ( P < .05). Conclusions Astrocytes have been shown to preserve neuronal viability via release of neurotrophic factors. Dexmedetomidine increases glial cell–derived neurotrophic factor from spinal cord astrocytes via the alpha-2 receptor. Treatment with alpha-2 agonist dexmedetomidine may be a clinical tool for use in spinal cord protection in aortic surgery.
Background Paraplegia remains a devastating complication of aortic surgery, occurring in up to 20% of complex thoracoabdominal repairs. Erythropoietin (EPO) attenuates this injury in models of spinal ...cord ischemia. Upregulation of the beta-common receptor (βcR) subunit of the EPO receptor is associated with reduced damage in murine models of neural injury. This receptor activates anti-apoptotic pathways including signaling transducer and activator of transcription 3 (STAT3). We hypothesized that spinal cord ischemia-reperfusion injury upregulates the βcR subunit with a subsequent increase in activated STAT3. Methods Adult male C57/BL6 mice received an intraperitoneal injection of 0.5 mL of EPO (10 U/kg) or 0.9% saline after induction of anesthesia. Spinal cord ischemia was induced through sternotomy and 4-minute thoracic aortic cross-clamp. Sham mice underwent sternotomy without cross-clamp placement. Four groups were studied: ischemic and sham groups, each with and without EPO treatment. After 4 hours of reperfusion, spinal cords were harvested and homogenized. The βcR subunit expression and STAT3 activation were evaluated by immunoblot. Results Ischemia reperfusion increased βcR subunit expression in spinal cords of ischemia + saline and ischemia + EPO mice compared with shams (3.4 ± 1.39 vs 1.31 ± 0.3, p = 0.01 and 3.80 ± 0.58 vs 1.56 ± 0.32, p = 0.01). Additionally, both ischemic groups demonstrated increased STAT3 activation compared with shams (1.35 ± 0.14 vs 1.09 ± 0.07, p = 0.01 and 1.66 ± 0.35 vs 1.08 ± 0.17, p = 0.02). Conclusions Ischemia-reperfusion injury induces EPO receptor βcR subunit expression and early downstream anti-apoptotic signaling through STAT3 activation. Further investigation into the role of the βcR subunit is warranted to determine tissue protective functions of EPO. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia.
Background Lung cancer stem cells (CSCs) are a subpopulation of cells that drive growth, invasiveness, and resistance to therapy. Inflammatory eicosanoids are critical to maintain this malignant ...subpopulation. Secretory phospholipase A2 group IIa (sPLA2 ) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA2 plays a role in the maintenance of lung CSCs. Methods Cancer stem cells from lung adenocarcinoma cell lines H125 and A549 were isolated using aldehyde dehydrogenase activity and flow cytometry. Protein and mRNA levels for sPLA2 were compared between sorted cells using Western blotting and quantitative reverse transcriptase–polymerase chain reaction techniques. Chemical inhibition of sPLA2 and short-hairpin RNA knockdown of sPLA2 were used to evaluate effects on tumorsphere formation. Results Lung CSCs were isolated in 8.9% ± 4.1% (mean ± SD) and 4.1% ± 1.6% of H125 and A549 cells respectively. Both sPLA2 protein and mRNA expression were significantly elevated in the CSC subpopulation of H125 ( p = 0.002) and A549 ( p = 0.005; n = 4). Knockdown of sPLA2 significantly reduced tumorsphere formation in H125 ( p = 0.026) and A549 ( p = 0.001; n = 3). Chemical inhibition of sPLA2 resulted in dose-dependent reduction in tumorsphere formation in H125 ( p = 0.003) and A549 ( p = 0.076; n = 3). Conclusions Lung CSCs express higher levels of sPLA2 than the non–stem cell population. Our findings that viral knockdown and chemical inhibition of sPLA2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA2 plays an important role in CSCs. These findings suggest that sPLA2 may be an important therapeutic target for human lung cancer.
Background The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, ...provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC. Study Design Rats were infused with doses of HBOC or saline through femoral vein injection (n = 5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response. Results Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific. Conclusions This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs.
Objectives Our aim was to determine whether aortic valve interstitial cells (AVICs) and pulmonary valve interstitial cells (PVICs) differ in expression of toll-like receptor (TLR)2 and TLR4, response ...to TLR agonists, and osteogenic phenotypic changes. Background Calcific stenosis occurs frequently in aortic valves but rarely in pulmonary valves. Studies have implicated AVICs in the inflammation associated with calcification and progression to stenosis. We previously reported that human AVICs express functional TLR2 and TLR4 and that stimulation of these receptors induces pro-osteogenic factor expression. Methods Human aortic and pulmonary valve leaflets from the same heart were collected and interstitial cells isolated. Results Aortic valves express more TLR2 and TLR4, in both tissue and isolated interstitial cells, than pulmonary valves. After stimulation with TLR2 and TLR4 agonists, AVICs express higher levels of pro-inflammatory and pro-osteogenic mediators (bone morphogenetic protein BMP-2, runt-related transcription factor 2) and greater osteogenic phenotypic changes (alkaline phosphatase ALP activity, calcified nodule formation) than PVICs. Silencing TLR2 and TLR4 in AVICs reduced BMP-2 expression and ALP activity to PVIC levels. ALP activity in AVICs induced by TLR2 and TLR4 agonists was abolished by BMP antagonism with Noggin and mimicked by stimulation with recombinant BMP-2. AVICs isolated from stenotic valves had greater expression of TLR2 and TLR4 and a greater BMP-2 response than AVICs from normal valves. Conclusions Greater expression of TLR2 and TLR4 and greater pro-inflammatory and pro-osteogenic responses to TLR2 and TLR4 agonists in AVICs than PVICs are associated with osteogenic phenotypic changes. These innate immune receptors may play a critical role in aortic valve calcification and stenosis.
Objective Group IIa secretory phospholipase A2 (sPLA2 IIa) plays a role in the malignant potential of several epithelial cancers. Nuclear factor kappa B (NF-κB) regulates cancer cell growth and is ...modulated by phospholipase activity in many cancer cells. We hypothesized that knockdown of sPLA2 in lung cancer cells would reduce cell proliferation and NF-κB activity in vitro and attenuate tumor growth in vivo. Methods Two human non–small cell lung cancer cell lines (A549 and H358) were transduced with short hairpin RNA targeting sPLA2 group IIa. Quantitative reverse transcriptase-polymerase chain reaction and immunoblotting confirmed knockdown of sPLA2 IIa messenger RNA and protein, respectively. Cell proliferation was evaluated by the 5-bromo-2′-deoxyuridine DNA labeling assay. NF-κB phosphorylation was assayed by western blot. 1 × 106 of A549 or A549 sPLA2 knockdown cells were injected into the left flanks of nude mice (aged 6 to 8 weeks). Tumors were followed for 23 days, then removed and stained with hematoxylin and eosin, stained with Ki-67, and analyzed for sPLA2 IIa messenger RNA expression. Results sPLA2 knockdown reduced NF-κB phosphorylation and tumor growth in vivo. A549 wild-type tumors grew twice as fast as knockdown tumors. Ki-67 staining was more prominent throughout the wild-type tumors compared with knockdown tumors. Explanted knockdown tumors maintained lower sPLA2 levels compared with wild-type, confirmed by reverse transcriptase-polymerase chain reaction. Conclusions Knockdown of sPLA2 IIa suppresses lung cancer growth in part by attenuating NF-κB activity. These findings justify further investigation into the cellular mechanisms of sPLA2 in lung cancer and its potential role as a therapeutic target.
Objective Bone morphogenic protein 2 is found in calcified areas of stenotic aortic valves, and prolonged stimulation of aortic valve interstitial cells with bone morphogenic protein 2 results in ...increased expression of alkaline phosphatase, indicating a mechanistic role for bone morphogenic protein 2 in aortic valve calcification. The purposes of this study were to assess the effect of bone morphogenic protein 2 on the expression of the osteogenic factors Runx2 and osteopontin in human aortic valve interstitial cells and to determine the signaling mechanisms that mediate the expression of these early osteogenic factors. Methods Interstitial cells were isolated from normal and stenotic human aortic valve leaflets, and cellular bone morphogenic protein 2 levels were analyzed by means of immunoblotting. Cultured interstitial cells from normal aortic valves were stimulated with bone morphogenic protein 2 to determine its effect on cellular Runx2 and osteopontin levels. Results Interstitial cells from stenotic aortic valves express greater levels of bone morphogenic protein 2 than cells from normal valves. Stimulation of human aortic valve interstitial cells with bone morphogenic protein 2 induced marked increases in Runx2 and osteopontin levels at 48 hours. The changes in Runx2 and osteopontin levels were preceded by phosphorylation of Smad1 and extracellular signal-regulated kinase 1/2 but not p38 mitogen-activated protein kinase. Silencing Smad1 reduced Runx2 and osteopontin levels, whereas inhibition of extracellular signal-regulated kinase 1/2 reduced osteopontin expression without an influence on Runx2 expression. Conclusions Interstitial cells of stenotic human aortic valves are characterized by increased bone morphogenic protein 2 levels. A short period of exposure of human aortic valve interstitial cells to bone morphogenic protein 2 induces the expression of Runx2 and osteopontin. The extracellular signal-regulated kinase 1/2 pathway modulates bone morphogenic protein 2–induced osteopontin expression, and the Smad1 pathway plays a role in regulating the expression of both Runx2 and osteopontin induced by bone morphogenic protein 2.
Abstract Background Overproduction of extracellular matrix (ECM) protein by aortic valve interstitial cells (AVICs) plays an important role in valvular sclerosis (thickening) associated with the ...early pathobiology of aortic stenosis. Accumulation of oxidized low-density lipoprotein (oxLDL) is observed in sclerotic aortic valve and may have a mechanistic role in valvular disease progression. Lysophosphatidylcholine (LysoPC) is a component of oxLDL and has multiple biological activities. This study was to test the hypothesis that oxLDL and LysoPC upregulate ECM protein production in human AVICs. Methods and results AVICs were isolated from normal human aortic valves. Cells were treated with oxLDL (40 μg/mL) or LysoPC (40 μmol/L). Immunoblotting was applied to analyze ECM proteins (collagens I and III and biglycan) in cell lysate and Picrosirius red staining is used to examine collagen deposition. Both oxLDL and LysoPC upregulated the production of biglycan and collagen I. The upregulation of ECM proteins by LysoPC was preceded by the phosphorylation of Akt and ERK1/2. Inhibition of Akt markedly reduced the effect of LysoPC on ECM protein production and collagen deposition. However, inhibition of ERK1/2 had no effect. Conclusions LysoPC upregulates the production of biglycan and collagen I in human AVICs and may mediate the effect of oxLDL on ECM protein production. The Akt pathway appears to be critical in mediating the effect of LysoPC. oxLDL accumulation and generation of LysoPC in the aortic valve tissue may contribute to the mechanism of valvular sclerosis associated with the development and progression of aortic stenosis.
Radiation induces osteogenesis in human aortic valve interstitial cells Nadlonek, Nicole A., MD; Weyant, Michael J., MD; Yu, Jessica A., MD ...
Journal of thoracic and cardiovascular surgery/The Journal of thoracic and cardiovascular surgery/The journal of thoracic and cardiovascular surgery,
12/2012, Letnik:
144, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Objective Irradiation of the chest or chest wall has been shown to cause calcific aortic stenosis. However, the mechanisms are unknown. Aortic valve interstitial cells have been implicated in the ...pathogenesis of aortic stenosis; they have been shown to change from the phenotype of a myofibroblast to an osteoblastlike cell. We therefore hypothesized that irradiation of human aortic valve interstitial cells induces an osteogenic phenotype. In isolated human aortic valve interstitial cells, our purpose was to determine the effect of irradiation on the production of osteogenic factors: (1) bone morphogenetic protein 2, (2) osteopontin, (3) alkaline phosphatase, and (4) the transcription factor Runx2. Methods Human aortic valve interstitial cells were isolated from normal aortic valves obtained from explanted hearts of patients undergoing cardiac transplantation (n = 4) and were grown in culture. The cells were grown to confluence, irradiated with 10 Gy using a cesium-137 irradiator, and then lysed 24 hours after irradiation. Cell lysates were analyzed via immunoblot and densitometry for bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. Statistical analysis was performed using analysis of variance, with P < .05 indicating significance. Results Irradiation induced an osteogenic phenotype in human aortic valve interstitial cells. Irradiation induced a 2-fold increase in bone morphogenetic protein 2, a 7-fold increase in osteopontin, a 3-fold increase in alkaline phosphatase, and a 2-fold increase in Runx2. Conclusions Radiation induces an osteogenic phenotype in human aortic valve interstitial cells. The irradiated cells had a significantly increased expression of the osteogenic factors bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. These data offer mechanistic insight into the pathogenesis of radiation-induced valvular heart disease.
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