Right Coronary Artery with an Intramural and Interarterial Course as a Unique Cause of Myocardial Ischemia: The Unroofing Method Might Still Be the Best Solution.

A 39-year-old man was admitted because of cardiac arrest. Emergent coronary angiography revealed a preserved coronary blood flow; however, multiple-row detector computed tomography (MDCT) revealed that the proximal right coronary artery (RCA) was running inside the aortic wall, creating proximal stenosis without atherosclerotic changes. Surgical intervention with unroofing was performed; however, postoperative stenosis of the proximal RCA required additional coronary artery bypass grafting (CABG). Intraoperative findings during CABG did not reveal hematoma or coronary dissection. However, MDCT one year after CABG depicted improvement of the RCA and graft stenoses, suggesting that the post-unroof stenosis may have been caused by an inflammatory reaction after surgical intervention.

Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux.

Atherosclerosis is a chronic inflammatory condition in which macrophages play a major role. Janus kinase 2 (JAK2) is a pivotal molecule in inflammatory and metabolic signaling, and Jak2V617F activating mutation has recently been implicated with enhancing clonal hematopoiesis and atherosclerosis. To determine the essential in vivo role of macrophage (M)-Jak2 in atherosclerosis, we generate atherosclerosis-prone ApoE-null mice deficient in M-Jak2. Contrary to our expectation, these mice exhibit increased plaque burden with no differences in macrophage proliferation, recruitment or bone marrow clonal expansion. Notably, M-Jak2-deficient bone marrow derived macrophages show a significant defect in cholesterol efflux. Pharmacologic JAK2 inhibition with ruxolitinib also leads to defects in cholesterol efflux and accelerates atherosclerosis. Liver X receptor agonist abolishes the efflux defect and attenuates the accelerated atherosclerosis that occurs with M-Jak2 deficiency. Macrophages of individuals with the Jak2V617F mutation show increased efflux which is normalized when treated with a JAK2 inhibitor. Together, M-Jak2-deficiency leads to accelerated atherosclerosis primarily through defects in cholesterol efflux from macrophages.

Role of myeloid-derived chemokine CCL5/RANTES at an early stage of atherosclerosis.

One of the hallmarks of atherosclerosis is ongoing accumulation of macrophages in the artery intima beginning at disease onset. Monocyte recruitment contributes to increasing macrophage abundance at early stages of atherosclerosis. Although the chemokine CCL5 (RANTES) has been studied in atherosclerosis, its role in the recruitment of monocytes to early lesions has not been elucidated. We show that expression of Ccl5 mRNA, as well as other ligands of the CCR5 receptor (Ccl3 and Ccl4), is induced in the aortic intima of Ldlr-/- mice 3 weeks after the initiation of cholesterol-rich diet (CRD)-induced hypercholesterolemia. En face immunostaining revealed that CCL5 protein expression is also upregulated at 3 weeks of CRD. Blockade of CCR5 significantly reduced monocyte recruitment to 3-week lesions, suggesting that chemokine signaling through CCR5 is critical. However, we observed that Ccl5-deficiency had no effect on early lesion formation and CCL5-blockade did not affect monocyte recruitment in Ldlr-/- mice. Immunostaining of the lesions in Ldlr-/- mice and reciprocal bone marrow transplantation (BMT) of Ccl5+/+ and Ccl5-/- mice revealed that CCL5 is expressed by both myeloid and endothelial cells. BMT experiments were carried out to determine if CCL5 produced by distinct cells has functions that may be concealed in Ccl5-/-Ldlr-/- mice. We found that hematopoietic cell-derived CCL5 regulates monocyte recruitment and the abundance of intimal macrophages in 3-week lesions of Ldlr-/- mice but plays a minor role in 6-week lesions. Our findings suggest that there is a short window in early lesion formation during which myeloid cell-derived CCL5 has a critical role in monocyte recruitment and macrophage abundance.

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

RATIONALE: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. OBJECTIVE: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. METHODS AND RESULTS: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT. CONCLUSIONS: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. CONCLUSIONS: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

Suppression of abdominal aortic aneurysm formation by AR-R17779, an agonist for the α7 nicotinic acetylcholine receptor.

OBJECTIVE: Activation of vagal nerve suppresses inflammatory responses through activation of α7 nicotinic acetylcholine receptor (nAchR). We sought to determine whether AR-R17779, a selective agonist of α7nAchR, affects the development of abdominal aortic aneurysm (AAA). METHODS AND RESULTS: AAA was induced by topical application of calcium chloride (CaCl2) to abdominal aorta (AAA group). NaCl (0.9%) was substituted for CaCl2 as a sham operation (SHAM group). AR-R17779 was administered in drinking water (AAA/AR-R group). One and 6 weeks after the operation, aortic tissue was excised for histological and molecular analyses. Aortic diameter and macrophage infiltration into the aortic adventitia were increased in AAA group compared with SHAM group at 6 weeks. Treatment with AR-R17779 reduced the diameter of the aorta and macrophage infiltration compared with AAA group. Wavy morphology of the elastic lamellae was lost in AAA group while it was preserved in AAA/AR-R group. Expression of inflammatory cytokines and matrix metalloproteinase (MMP) activities were enhanced in AAA group, which was suppressed in AAA/AR-R group. AR-R17779 treatment suppressed CaCl2-induced expression of cytokines, activities of MMPs and NF-κB activation at 1 week when aortic dilatation had not developed. CONCLUSION: Treatment with AR-R17779 prevented the enlargement of abdominal aorta induced by CaCl2 in association with reduced inflammation and extracellular matrix disruption. These findings suggest therapeutic potential of α7nAchR activation for prevention of AAA development.

PPARγ Agonists Attenuate Palmitate-Induced ER Stress through Up-Regulation of SCD-1 in Macrophages.

BACKGROUND: Clinical trials have shown that treatment of patients with type 2 diabetes with pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, reduces cardiovascular events. However, the effect of PPARγ agonists on endoplasmic reticulum (ER) stress that plays an important role in the progression of atherosclerosis has not been determined. We sought to determine the effect of PPARγ agonists on ER stress induced by palmitate, the most abundant saturated fatty acid in the serum. METHODS AND RESULTS: Protein expression of ER stress marker was evaluated by Western blot analysis and stearoyl-CoA desaturase1 (SCD-1) mRNA expression was evaluated by qRT-PCR. Macrophage apoptosis was detected by flowcytometry. Pioglitazone and rosiglitazone reduced palmitate-induced phosphorylation of PERK, a marker of ER stress, in RAW264.7, a murine macrophage cell line. Pioglitazone also suppressed palmitate-induced apoptosis in association with inhibition of CHOP expression, JNK phosphorylation and cleavage of caspase-3. These effects of pioglitazone were reversed by GW9662, a PPARγ antagonist, indicating that PPARγ is involved in this process. PPARγ agonists increased expression of SCD-1 that introduces a double bond on the acyl chain of long-chain fatty acid. 4-(2-Chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide, an inhibitor of SCD-1, abolished the anti-ER stress and anti-apoptotic effects of pioglitazone. These results suggest that PPARγ agonists attenuate palmitate-induced ER stress and apoptosis through SCD-1 induction. Up-regulation of SCD-1 may contribute to the reduction of cardiovascular events by treatment with PPARγ agonists.

Stimulation of α7 nicotinic acetylcholine receptor by AR-R17779 suppresses atherosclerosis and aortic aneurysm formation in apolipoprotein E-deficient mice.

Atherosclerosis is a chronic inflammatory disease. It has been appreciated that vagus nerve inhibits macrophage activation via α7 nicotinic acetylcholine receptor (nAChR), termed the cholinergic anti-inflammatory pathway. We explored the effects of AR-R17779, a selective α7nAChR agonist, on atherosclerosis and aneurysm formation in apolipoprotein E (ApoE)-deficient mice. ApoE-deficient mice were fed a high-fat diet (HFD) and angiotensin II (Ang II) was infused by osmotic minipumps from 10-week-old for 4weeks. AR-R17779 was given in drinking water ad libitum. Oil red O staining of the aorta showed that combined loading of HFD and Ang II induced marked atherosclerosis compared with control mice fed a normal chow. Treatment with AR-R17779 significantly reduced atherosclerotic plaque area and improved survival of mice. Treatment with AR-R17779 also suppressed abdominal aortic aneurysm formation. Quantitative RT-PCR of the aorta revealed that mRNA expression levels of interleukin-1ß, interleukin-6 and NOX2 were significantly decreased in AR-R17779-treated mice compared with Ang II+HFD mice. AR-R17779 treatment also reduced blood pressure and serum lipid levels. In conclusion, α7nAChR activation attenuates atherogenesis and aortic abdominal aneurysm formation in ApoE-deficient mice possibly through an anti-inflammatory effect and reduction of blood pressure and lipid levels. Pharmacological activation of α7nAChR may have a therapeutic potential against atherosclerotic vascular diseases through multiple mechanisms.

Suppression of abdominal aortic aneurysm formation by inhibition of prolyl hydroxylase domain protein through attenuation of inflammation and extracellular matrix disruption.

In the present study we sought to determine the effect of CoCl2, an inhibitor of PHD (prolyl hydroxylase domain protein), on the development of AAA (abdominal aortic aneurysm). AAA was induced in C57BL/6 mice by periaortic application of CaCl2 (AAA group). NaCl (0.9%)-treated mice were used as a sham control (SHAM group). Mice were treated with 0.05% CoCl2 in the drinking water (AAA/CoCl2 group). At 1 and 6 weeks after the operation, aortic tissue was excised for further examination. After 6 weeks of CaCl2 treatment, aortic diameter and macrophage infiltration into the aortic adventitia were increased in the AAA group compared with the SHAM group. Treatment with CoCl2 reduced the aneurysmal size and macrophage infiltration compared with the AAA group. Aortic expression of inflammatory cytokines and MCP-1 (monocyte chemoattractant protein-1) and the activities of MMP-9 (matrix metalloproteinase-9) and MMP-2 were enhanced in the AAA group and attenuated in the AAA/CoCl2 group. Expression of cytokines and the activities of MMPs were already increased after 1 week of CaCl2 treatment, but were suppressed by CoCl2 treatment in association with reduced NF-κB (nuclear factor κB) phosphorylation. Treatment with CoCl2 in mice prevented the development of CaCl2-induced AAA in association with reduced inflammation and ECM (extracellular matrix) disruption. The results of the present study suggest that PHD plays a critical role in the development of AAA and that there is a therapeutic potential for PHD inhibitors in the prevention of AAA development.

Deletion of phd2 in myeloid lineage attenuates hypertensive cardiovascular remodeling.

BACKGROUND: Hypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia-inducible factor-α (HIF-α) and thereby induces HIF-α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid-specific Phd2 deletion affects hypertension-induced cardiovascular remodeling. METHODS AND RESULTS: Myeloid-specific PHD2-deficient mice (MyPHD2KO) were generated by crossing Phd2-floxed mice with LysM-Cre transgenic mice, resulting in the accumulation of HIF-1α and HIF-2α in macrophage. Eight- to ten-week-old mice were given N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L-NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor-ß and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L-NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF-α synthesis to L-NAME/Ang II-treated MyPHD2KO mice reversed these beneficial features. CONCLUSIONS: Phd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation- and fibrosis-associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.

Prolyl hydroxylase domain protein 2 plays a critical role in diet-induced obesity and glucose intolerance.

BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.

Beraprost sodium, a stable prostacyclin analogue, improves insulin resistance in high-fat diet-induced obese mice.

Obesity induces hypertrophy of adipocyte resulting in production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1 (CCL2)). These cytokines play an important role in the development of insulin resistance. Beraprost sodium (BPS), a prostaglandin I2 analogue, is reported to attenuate inflammation. In this study, we examined the effect of BPS on glucose metabolism in mice fed a high-fat diet (HFD). Four-week-old C57/B6 male mice were fed a HFD for 12 weeks (HFD group) and the treatment group received oral BPS (300 µg/kg per day) for the same period. Then, glucose metabolism, histological changes, and gene expression of white adipose tissue (WAT) were examined. Body weight was increased, and glucose intolerance and insulin resistance were developed in the HFD group. Treatment with BPS improved glucose tolerance and insulin action without body weight change. Histological analysis of WAT showed an increase in the size of adipocyte and macrophage infiltration in the HFD group, which was attenuated by BPS treatment. BPS reduced HFD-induced expression of MCP1 and TNF-α in WAT. BPS also attenuated hepatic steatosis induced by the HFD. These results suggest that BPS improved glucose intolerance possibly through suppression of inflammatory cytokines in WAT. BPS may be beneficial for the treatment of obesity-associated glucose intolerance.

Acetylcholinesterase inhibitors attenuate angiogenesis.

Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimer's disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to determine whether donepezil affects angiogenesis. A hindlimb ischaemia model was created by unilateral femoral artery ligation. Blood flow recovery examined by laser Doppler perfusion imaging and capillary density by immunohistochemical staining of CD31-positive cells in the ischaemic hindlimb were significantly decreased in donepezil- and physostigmine-treated mice compared with control mice after 2 weeks. Donepezil reduced expression of IL (interleukin)-1ß and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1ß to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1ß expression in C2C12 myoblast cells, which was inhibited by pre-incubation with ACh (acetylcholine) or LY294002, a PI3K (phosphoinositide 3-kinase) inhibitor. Donepezil inhibited phosphorylation of Akt [also known as PKB (protein kinase B)], a downstream kinase of PI3K, in the ischaemic hindlimb. These findings suggest that cholinergic stimulation by acetylcholinesterase inhibitors suppresses angiogenesis through inhibition of PI3K-mediated IL-1ß induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.

Inhibition of prolyl hydroxylase domain-containing protein downregulates vascular angiotensin II type 1 receptor.

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation.

AIMS: Tumour protein p53 plays an important role in the vascular remodelling process as well as in oncogenesis. p53 is negatively regulated by murine double minute 2 (MDM2). A recently developed MDM2 inhibitor, nutlin-3, is a non-genotoxic activator of the p53 pathway. So far, the effect of MDM2 inhibition on vascular remodelling has not been elucidated. We therefore investigated the effect of nutlin-3 on neointima formation. METHODS AND RESULTS: Nutlin-3 up-regulated p53 and its downstream target p21 in vascular smooth muscle cells (VSMCs). DNA synthesis assay and flow cytometric analysis revealed that nutlin-3 inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation by cell cycle arrest. This inhibitory effect was abrogated in p53-siRNA-transfected VSMCs. Furthermore, nutlin-3 inhibited PDGF-stimulated VSMC migration. Treatment with nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury in mice, associated with up-regulation of p53 and p21. BrdU incorporation was decreased at 14 days after injury in nutlin-3-treated mice. TUNEL assay showed that nutlin-3 did not exaggerate apoptosis of the injured vessels. Infiltration of macrophages and T-lymphocytes and mRNA expression of chemokine (C-C motif) ligand-5, interleukin-6, and intercellular adhesion molecule-1 were decreased in the injured vessels of nutlin-3-treated mice. Nutlin-3 suppressed NF-κB activation in VSMCs, but not in p53-siRNA-transfected VSMCs. CONCLUSIONS: The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Inhibition of prolyl hydroxylase domain-containing protein suppressed lipopolysaccharide-induced TNF-alpha expression.

OBJECTIVE: Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of alpha-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) induction in macrophages. METHODS AND RESULTS: Northern blot analysis and ELISA revealed that LPS-induced TNF-alpha upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-alpha upregulation in HIF-1alpha-depleted cells and HIF-1alpha overexpression failed to suppress the induction of TNF-alpha. DMOG rather suppressed LPS-induced NF-kappaB transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-alpha induction. DMOG also inhibited LPS-induced TNF-alpha production in peritoneal macrophages as well as human macrophages. CONCLUSIONS: PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-alpha upregulation in macrophages possibly through NF-kappaB inhibition, which is independent of HIF-1alpha accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases.

Correlation of decreased survival and IL-18 in bone metastasis.

OBJECTIVE: Previous studies have reported that serum IL-18 levels are increased in some cancers. We investigated whether IL-18 production is increased in sera and cancer cells of patients with non-small cell lung cancer (NSCLC). PATIENTS OR MATERIALS: Serum levels of IFN-gamma and IL-18 and thioredoxin 1 (TRX1) were measured in 79 patients (51 males, 28 females, median age 67 years) with advanced NSCLC (57 adenocarcinoma, 22 squamous cell carcinoma; TNM stages IIIA [n=11], IIIB [n=24], and IV [n=44]) and 75 healthy age-matched controls (44 males, 31 females, median age 65 years) by enzyme-linked immunosorbent assay. We examined IL-18 production in the lungs and sites of bone metastasis of adenocarcinoma by immunohistochemistry. RESULTS: Serum IL-18, IFN-gamma, and TRX1 levels in NSCLC patients were significantly (p<0.0001, p=0.0031, and p<0.0001, respectively) higher than in control subjects, while serum IFN-gamma levels in NSCLC were slightly increased. Serum IL-18, but not IFN-gamma or TRX1, levels were significantly (p=0.0102) and negatively associated with overall survival in NSCLC. The serum IL-18 level was identified as an independent prognostic factor for overall survival in multivariate survival analysis. Moreover, serum IL-18 levels were significantly (p=0.049) higher in NSCLC with bone metastasis than in NSCLC without bone metastasis. Based on immunohistochemistry, we observed that cancer cells in the lungs and bone metastases markedly produced IL-18. CONCLUSION: Our results suggest that elevated serum IL-18 levels may be associated with IL-18 producing cancer cells in advanced NSCLC.

Expression of ERCC1 and class III beta-tubulin in non-small cell lung cancer patients treated with a combination of cisplatin/docetaxel and concurrent thoracic irradiation.

INTRODUCTION: The expression of excision repair cross-complementation group 1 (ERCC1) is reported to be correlated with resistance to platinum-based drugs. Class III beta-tubulin is reported to be correlated with resistance to taxanes. METHODS: In the present study, we evaluated whether ERCC1 and class III beta-tubulin expression could be used to predict progression-free and/or overall survival in 34 patients with locally advanced non-small cell lung cancer (NSCLC) receiving concurrent chemoradiation therapy with cisplatin and docetaxel, and immunohistochemistry was used to examine the expression of these two proteins in tumor samples obtained from the patients. RESULTS: Immunostaining for ERCC1 and class III beta-tubulin was positive in 16 and 12 patients, respectively. A significant correlation was observed between ERCC1 expression and response to chemotherapy (P = 0.012), and between class III beta-tubulin expression and histology (P = 0.029). Patients negative for ERCC1 had a significantly longer median progression-free (62.5 vs. 36 weeks, P = 0.009), but not overall (171 vs. 50.5 weeks, P = 0.208), survival than those positive for ERCC1. Expression of class III beta-tubulin was not correlated with progression-free or overall survival (P = 0.563 and P = 0.265, respectively). Multivariate analysis adjusting for possible confounding factors showed that negative ERCC1 expression (hazard ratio = 3.972, P = 0.009) was a significantly favorable factor for progression-free survival. CONCLUSIONS: This retrospective study indicates that immunostaining for ERCC1 may be useful for predicting survival in NSCLC patients receiving concurrent chemoradiotherapy with cisplatin and docetaxel, and can provide information critical for planning personalized chemotherapy.

Expression of ERCC1 and class III beta-tubulin in non-small cell lung cancer patients treated with carboplatin and paclitaxel.

The combination of carboplatin and paclitaxel is the most commonly used regimen for the treatment of advanced non-small cell lung cancer (NSCLC) patients. The expression of excision repair cross-complementation group 1 (ERCC1) is reported to be correlated with resistance to platinum-based drugs. Class III beta-tubulin is reported to be correlated with resistance to taxanes. We evaluated whether ERCC1 and class III beta-tubulin expression could predict progression-free and/or overall survival in relapsed NSCLC patients treated with carboplatin and paclitaxel. Immunohistochemistry was used to examine the expression of these two proteins in resected lung tumor samples obtained from 45 patients treated with carboplatin and paclitaxel against recurrent tumors after curative resection. Immunostaining for ERCC1 and class III beta-tubulin was positive in 20 and 16 patients, respectively. Patients negative for ERCC1 had a significantly longer median progression-free (44 weeks vs. 28 weeks, P=0.046) and overall (102 weeks vs. 56 weeks, P=0.010) survival than those positive for ERCC1. Patients negative for class III beta-tubulin expression had a significantly longer median progression-free (40 weeks vs. 35 weeks, P=0.031), but not overall (78 weeks vs. 57 weeks, P=0.087), survival than those positive for class III beta-tubulin expression. In particular, patients negative for both ERCC1 and class III beta-tubulin had significantly longer progression-free (P=0.036) and overall survival (P=0.015), compared with those positive for ERCC1 and/or class III beta-tubulin. In multivariate analysis, negative class III beta-tubulin expression (hazard ratio=1.912, P=0.048) was significantly favorable factor for progression-free survival, and negative ERCC1 expression (hazard ratio=2.580, P=0.014) and better performance status (hazard ratio=3.287, P=0.007) were significantly favorable factors for overall survival. This retrospective study indicates that immunostaining for ERCC1 and class III beta-tubulin may be useful for predicting survival in NSCLC patients receiving carboplatin and paclitaxel against recurrent tumors after curative resection and can provide information critical for planning personalized chemotherapy.