Mendelian randomization analysis reveals causal relationships between circulating cell traits and renal disorders.

Purpose: The aim of this study was to investigate the causal relationships between circulating cell traits and risk of renal disorders. Methods: We applied a comprehensive two-sample Mendelian randomization (MR) analysis. Single nucleotide polymorphisms (SNPs) from publicly available genome-wide association studies (GWAS) databases were utilized. Genetically predicted instrumental variables of human blood cell traits were extracted from Blood Cell Consortium (BCX) while data on renal diseases was obtained from Finngen consortium. The primary MR analysis was conducted using the inverse variance weighted (IVW) method, with the weighted median (WM) and MR-Egger models used as additional methods. Sensitivity analyses, including MR-PRESSO, radial regression and MR-Egger intercept were conducted to detect outliers and assess horizontal pleiotropy. We further utilized the leave-one-out analysis to assess the robustness of the results. Causal associations were considered significant based on false rate correction (FDR), specifically when the IVW method provided a pFDR < 0.05. Results: Our results demonstrated that both white blood cell (WBC) count (OR = 1.50, 95% CI = 1.10-2.06, pFDR = 0.033, pIVW = 0.011) and lymphocyte count (OR = 1.50, 95% CI = 1.13-1.98, pFDR = 0.027, pIVW = 0.005) were causally associated with a higher risk of IgA nephropathy. Furthermore, WBC count was identified as a significant genetic risk factor for renal malignant neoplasms (OR = 1.23, 95% CI = 1.06-1.43, pFDR = 0.041, pIVW = 0.007). Additionally, an increased level of genetically predicted eosinophils was found to be causally associated with a higher risk of diabetic nephropathy (OR = 1.21, 95% CI = 1.08-1.36, pFDR = 0.007, pIVW = 0.001). No evidence of pleiotropy was determined. Conclusion: Our findings provide evidence of causal associations of circulating WBC count, lymphocyte count and IgA nephropathy, WBC count and renal malignant neoplasms, and eosinophil count and diabetic nephropathy. These results have the potential to contribute to the development of novel diagnostic options and therapeutic strategies for renal disorders.

A network meta-analysis of the efficacy of hypoxia-inducible factor prolyl-hydroxylase inhibitors in dialysis chronic kidney disease.

BACKGROUND: Five types of HIF-PHIs have been authorized for anemia treatment in CKD patients in China and Japan. These are enarodustat, roxadustat, daprodustat, vadadustat, and molidustat. How effectively they compare to ESAs about clinical results in CKD-DD patients is uncertain. This study examined the RCT evidence about the benefits and risks of HIF-PHIs and ESAs in dialysis CKD patients. METHODS: We conducted an extensive investigation and network meta-analysis of RCTs. In these RCTs, patients with CKD-DD received one of five different HIF-PHI or ESAs, a placebo, and no medical intervention. Outcomes included hemoglobin, iron parameters, and adverse events, and there were four weeks of follow-up at least. A frequentist framework for multivariate random effects meta-analyzed the results. The effect sizes of categorical variables were displayed as odds ratios. Mean differences were employed for computing continuous outcomes with common units; otherwise, standardized mean differences were applied. The Cochrane tool evaluated the bias risk in RCTs. RESULTS: 26 RCTs with 14945 patients were qualified for inclusion. Compared to the placebo, HIF-PHIs and ESAs dramatically boosted hemoglobin without affecting serum iron. Roxadustat performed better hemoglobin levels than ESAs (MD 0.32, 95% CI 0.10 to 0.53) and daprodustat (0.46, 0.09 to 0.84). Roxadustat (91.8%) was the top hemoglobin treatment among all medical interventions, as determined by the SUCRA ranking. However, roxadustat caused more thrombosis and hypertension than ESAs (1.61, 1.22 to 2.12) and vadadustat (1.36, 1.01 to 1.82). The lowest rates of hypertension and thrombosis were seen in molidustat (80.7%) and ESAs (88.5%). Compared with a placebo, ESAs and HIF-PHIs all affected TSAT levels. Except for molidustat, the other four HIF-PHIs impact different iron parameters. Regarding ferritin reduction, roxadustat (90.9%) and daprodustat (60.9%) came out on top. Enarodustat (80.9%) and roxadustat (74%) placed best and second in lowering hepcidin levels. The former two medicines for TIBC improvement were vadadustat (98.7%) and enarodustat (80.9%). CONCLUSION: The most effective treatment for hemoglobin correction is roxadustat. The superior efficacy of reducing hepcidin makes roxadustat and enarodustat appropriate for patients with inflammation. However, the increased risk of hypertension and thrombosis associated with roxadustat should be noted. In patients at risk for hypertension and thrombosis, molidustat and ESAs may be preferable options. When administering roxadustat and daprodustat, clinicians should check ferritin to assess iron storage. Lower TSAT in patients receiving HIF-PHIs and ESAs treatment suggests intravenous iron supplements are needed.

Epidemiological characteristics and factors affecting blood pressure of migratory hypertensive population in Hainan Province A study of 180 cases / 中国热带医学

@#Abstract: Objective　To understand the epidemiological characteristics of the migratory bird population with hypertension in Hainan and to provide a basis for the health management of the migratory bird population. Methods　One hundred and eighty migratory birds with hypertension who had lived in in the Mangrove Bay community of Chengmai, Hainan for 3 months or more were selected as the study population. Demographic data, including gender, age, ethnicity, occupation, medication use, family history of hypertension, hometown living environment, Hainan living environment (presence of water area and greenbelt around the residence, type of water area and greenbelt), smoking and alcohol consumption, and height and weight were collected by questionnaire and physical examination to investigate the epidemiological characteristics of the study subjects, and unconditional dichotomous logistic regression analysis was used to identify factors affecting the reduction of blood pressure among the hypertensive migratory bird population before and after taking medication in Hainan. Results　A total of 180 study subjects were included, including 77 (42.8%) males and 103 (57.2%) females, with 78 (43.3%) subjects aged ≤65. The hypertension levels were: 87 (48.3%) had normal high blood pressure, 56 (31.1%) had grade 1 hypertension and 37 (20.6%) had grade 2 hypertension. The higher the blood pressure classification, the lower the percentage of migratory individuals whose blood pressure dropped during their stay in Hainan. Age, hypertension classification, dietary habits, living environment, body mass index (BMI), and alcohol consumption were associated with a decrease in blood pressure during Hainan among the study participants who took hypertension medication regularly. Salty diet (OR=2.778, 95%CI:1.070-7.213, P=0.036) and alcohol consumption (OR=2.686, 95%CI:1.042-6.925, P=0.041) were unfavorable factors for lower diastolic blood pressure before taking medication; overweight (OR=3.487, 95%CI:1.306-9.310, P=0.013) was an unfavorable factor in the reduction of diastolic blood pressure after taking medication. Conclusion　The blood pressure reduction is more significant in migratory individuals in migratory individuals under 65 years old, with a light diet, no alcohol consumption and normal BMI. Environmental factors, age, dietary habits and lifestyle are the main causes of blood pressure, which can provide a reference basis for the health management of the migratory birds with hypertension in Hainan.

Efficacy of brain natriuretic peptide vs. nicorandil in preventing contrast-induced nephropathy: a network meta-analysis.

This study aimed to conduct a network meta-analysis (NMA) to compare the efficacy of brain natriuretic peptide (BNP) vs nicorandil for preventing contrast-induced nephropathy (CIN). Databases of Pubmed, Cochrane, Embase, Web of Science were searched by keywords for eligible studies of randomized controlled trials investigating different agents (BNP, nicorandil, nitroglycerin, intravenous saline) for preventing CIN. The outcomes included a change in serum creatinine level at 48 h and the incidence of CIN after percutaneous coronary intervention (PCI) or coronary angiography (CAG). A total of 13 studies with 3,462 patients were included. Compared with intravenous saline alone, except for nitroglycerin (odds ratio [OR]: 1.02, 95% CI [0.36-2.88]), the other drugs significantly reduced the CIN incidence with OR of 0.35 (95% CI [0.24-0.51]) for BNP, 0.52 (0.29, 0.94) for usual-dose nicorandil, 0.28 (0.19, 0.43) for double-dose nicorandil. BNP and double-dose nicorandil significantly decreased the change of serum creatinine (SCr) levels with mean difference (MD) of -6.98, (-10.01, -3.95) for BNP, -8.78, (-11.63, -5.93) for double-dose nicorandil. No significant differences were observed in the change of SCr levels for nitroglycerin (-4.97, [-11.46, 1.52]) and usual-dose nicorandil (-2.32, [-5.52, 0.89]) compared with intravenous saline alone. For double-dose nicorandil, the CIN incidence and the change of SCr level in group of 4-5 days treatment course were more than group of less than or equal to 24 h treatment course (OR of 1.48, [0.63-3.46] and MD of 2.48, [-1.96, 6.91]). In conclusion, BNP and double-dose nicorandil can have effects on preventing the incidence of CIN and double-dose nicorandil performed better than BNP. In double-dose nicorandil groups, a course of less than or equal to 24 h before and after procedure performed with better efficacy than a course of 4-5 days.

Cancer survival in patients from a hospital-based cancer registry, China.

Purpose: There are few reports on survival rate analysis from hospital-based cancer registries (HBCR) in China, although the National Center of Cancer Registry of China has launched such an effort with the mission to expand the scope of registration and follow-up. Our study aimed to evaluate survival and outcomes of cancer patients from a HBCR in eastern China. Methods: Active and passive follow-up methods were used to obtain information on survival status for all patients from Qidong City and Haimen City in the databases of our hospital-based registrations from 2002 to 2014. Censor time for survival was 31st March, 2016. Survival probability was estimated using the life-table method with SPSS Statistics software, and comparison of significant differences in survival rates was tested by Wilcoxon (Gehan) statistic. Results: The outcomes of 5010 patients were identified in the follow-up for 5244 cases from Qidong and Haimen, with a follow-up rate of 95.65%, and a rate of lost to follow-up of 4.35%. The 1-, 3-, 5-, and 10-year observed survival (OS) rate in all-combined cancer sites were 59.80%, 37.70%, 30.82%, and 22.60%, respectively. The top 10 cancer sites in rank were cancers of lung, esophagus, liver, cervix, stomach, breast, colon-rectum, non-Hodgkin's lymphoma, nasopharynx, and ovary, with 5-year OS rates of 12.63%, 19.62%, 11.69%, 66.61%, 21.35%, 59.43%, 36.36%, 37.03%, 48.95% and 36.17%, respectively. Females experienced better survival than males for lung, esophageal, liver, nasopharyngeal and pancreatic cancers (P<0.05), but not for other sites (P>0.05). A significant difference was also found between males and females when all-sites were combined (P<0.01). There are significant differences (P<0.05) between the 2015 patients (from Qidong) and the 3001 patients (from Haimen) with 5-year OS rates of 32.72% vs 29.57%; no significant differences were found for 5-year OS rates for individual cancer sites (P>0.05) except for liver (P=0.0005) and ovary (P=0.0460) between the two cities. Younger patients had better prognosis, but significance was only seen in cervical (P=0.0102) and nasopharyngeal (P=0.0305) cancers. Conclusion: The survival rates of each site or of all sites-combined in this setting are consistent with those elsewhere in China and abroad. Discrepancies in overall survival could be affected by the proportion of sites with or without better prognosis. Hospital-based cancer survival is a better index to evaluate outcomes that reflect the levels of comprehensive treatment and improvement of medical and health services.

Cancer survival in Qidong between 1972 and 2011: A population-based analysis.

Population-based cancer survival is an improved index for evaluating the overall efficiency of cancer health services in a given region. The current study analysed the observed survival and relative survival of leading cancer sites from a population-based cancer registry between 1972 and 2011 in Qidong, China. A total of 92,780 incident cases with cancer were registered and followed-up for survival status. The main sites of the cancer types, based on the rank order of incidence, were the liver, stomach, lung, colon and rectum, oesophagus, breast, pancreas, leukaemia, brain and central nervous system (B and CNS), bladder, blood [non-Hodgkin's lymphoma (NHL)] and cervix. For all malignancies combined, the 5-year observed survival was 13.18% and the relative survival was 15.80%. Females had higher observed survival and relative survival (19.32 and 22.71%, respectively) compared with males (9.63 and 11.68%, respectively). The cancer sites with the highest five-year relative survival rates were the female breast, bladder, cervix and colon and rectum; followed by NHL, stomach, B and CNS cancer and leukaemia. The poorest survival rates were cancers of oesophagus, lung, pancreas and liver. Higher survival rates were observed in younger patients compared with older patients. Cancers of the oesophagus, female breast and bladder were associated with higher survival in middle-aged groups. Improved survival rates in the most recent two 5-year calendar periods were identified for stomach, lung, colon and rectum, oesophagus, female breast and bladder cancer, as well as leukaemia and NHL. The observations of the current study provide the opportunity for evaluation of the survival outcomes of frequent cancer sites that reflects the changes and improvement in a rural area in China.

IgG4-related Kidney Disease Mimicking Malignant Ureter Tumor: Case Report and Literature Review.

Immunoglobulin G4-related disease is a recently recognized systemic disease that can affect any organ or tissue in the body, including the kidneys. IgG4-related kidney disease (IgG4-RKD) is an important part of immunoglobulin G4-related disease. The most common renal manifestation of IgG4-RKD is tubulointerstitial nephritis and glomerular lesions. There, however, is few case of IgG4-RKD mimicking malignant ureter tumor leading to severe hydronephrosis. We herein report an unusual case of IgG4-RKD mimicking malignancy.A 66-year-old Asian man presented to the nephrologist with soreness of loins, anorexia, and acute kidney injury in 2010. His renal function spontaneously improved after 2 weeks' hemodialysis without systemic steroid therapy. Four years later, he presented to the urologist with severe left hydronephrosis because of marked thickness of the left ureter wall. As a ureteral malignancy could not be ruled out, laparoscopic nephroureterectomy was performed.IgG4-related kidney disease was confirmed by the histologic examination. Then, repeat laboratory test showed almost complete recovery of renal function after initiation of steroidal therapy.This case highlights the rare possibility of IgG4-RKD mimicking malignant ureter tumor. Nephrologist and pathologists should be aware of the possibility that hydronephrosis with ureter obstruction may be involved in IgG4-RKD.

Tubulointerstitial Nephritis and Uveitis Syndrome in an Elderly Man: Case Report and Literature Review.

Tubulointerstitial nephritis and uveitis (TINU) syndrome is a rare disease of unknown etiology defined by the combination of tubulointerstitial nephritis, uveitis, and biochemical abnormalities. It has been reported that TINU mainly affects adolescents and young women. Here we reported a special case regarding a 60-year-old man with acute renal failure due to TINU syndrome documented by renal biopsy.We present a rare case of an elderly patient, who had been suffering from a fever for 2 weeks, characterized by sudden onset and resolving spontaneously, and accompanied by extreme fatigue, loss of appetite, and shivering. Renal biopsy showed a tubulointerstitial nephritis, with polymorphonuclear infiltration and acute tubulitis. In the outpatient clinic, he was diagnosed with idiopathic bilateral anterior uveitis 1 month ago. Ophthalmological examination revealed anterior asymptomatic bilateral uveitis. Human leukocyte antigen (HLA) typing (HLA-DQA1*0101/0201 and HLA-DQB1*0303/0503) was found which supported the suspect of TINU syndrome. The patient was treated with oral prednisone (1 mg/kg) and continued for 8 weeks on tapering doses. Serum creatinine normalized within 3 and 6 months later renal function also recovered completely.This case highlights that TINU syndrome is probably an underdiagnosed disease responsible for some cases of idiopathic anterior uveitis in elderly male patients. It is of critical importance to be aware of this syndrome by nephrologist and ophthalmologists in this special population. Further studies are needed to elucidate clinical characteristic and pathogenesis of TINU syndrome in elderly population.

Di-(2-ethylhexyl) phthalate adjuvantly induces imbalanced humoral immunity in ovalbumin-sensitized BALB/c mice ascribing to T follicular helper cells hyperfunction.

Di-(2-ethylhexyl) phthalate (DEHP) has been considered as a widespread environmental persistent organic pollutant and its potential concern on human health is raised by previous studies. In particular, children are more likely to be exposed to DEHP through gastrointestinal route and consequently are more susceptible to DEHP hazards. Some reports have uncovered a positive association between DEHP exposure and an increased prevalence of allergic diseases in infants and juveniles. Allergy is a hypersensitive reaction rooted in imbalanced humoral immunity. T follicular helper cell (Tfh), an important CD4(+) Th cell subset, until recently has been identified as a key player in humoral immune response by modifying B cells functions. Tfh cells are therefore perceived as the therapeutic target of immune disorders. In the present study, focusing on the newly confirmed Tfh cells, we examined the effects of DEHP on humoral immunity and investigated the underlying mechanisms. Using ovalbumin (OVA) sensitization weanling mice model under the condition of gastrointestinal exposure to DEHP, we found that DEHP acted as an immunoadjuvant to augment OVA-specific IgE and IgG1 production, amplified germinal center formation in lymphoid nodule, as well as stimulated the expansion of CD4(+)CXCR5(+)ICOS(+)/CD4(+)CXCR5(+)PD-1(+) Tfh cells and CD19(+)CD138(+)GL7(+) plasma cells. Based on the results of immune adoptive transfusion, DEHP-related anaphylactic response was ascribed to Tfh cells hyperfunction directly. We further proved that DEHP gavage together with OVA sensitization adjuvantly promoted the synthesis of cytokines IL-21 and IL-4 and the expression of transcription factors Bcl-6 and c-Maf in Tfh cells. In conclusion, our study demonstrates that DEHP has adjuvant toxic effects on Tfh cells by synthesizing an excess of cytokines IL-21 and IL-4 via over-expression of transcription factors Bcl-6 and c-Maf, leading to an increasing secretion of allergy-related IgE and IgG1.

Proliferative and survival effects of PUMA promote angiogenesis.

The p53 upregulated modulator of apoptosis (PUMA) is known as an essential apoptosis inducer. Here, we report the seemingly paradoxical finding that PUMA is a proangiogenic factor critically required for the proliferation and survival of vascular and microglia cells. Strikingly, Puma deficiency by genetic deletion or small hairpin RNA knockdown inhibited developmental and pathological angiogenesis and reduced microglia numbers in vivo, whereas Puma gene delivery increased angiogenesis and cell survival. Mechanistically, we revealed that PUMA plays a critical role in regulating autophagy by modulating Erk activation and intracellular calcium level. Our findings revealed an unexpected function of PUMA in promoting angiogenesis and warrant more careful investigations into the therapeutic potential of PUMA in treating cancer and degenerative diseases.

MMP-2 and MMP-9 are prominent matrix metalloproteinases during atherosclerosis development in the Ldlr(-/-)Apob(100/100) mouse.

Matrix-degrading proteases capable of degrading components of the extracellular matrix may play an important role in development and progression of atherosclerotic lesions. In the present study, we used the Ldlr(-/-)Apob(100/100) mouse model, which has a plasma lipoprotein profile similar to that of humans with atherosclerosis, to study the expression of matrix metalloproteinases (MMPs) during early stages of atherosclerosis development. We analyzed the expression of 11 proteases and three protease inhibitors in 5- to 40-week-old Ldlr(-/-)Apob(100/100) mice. Expression and activity of MMP-2 and MMP-9 was increased in advanced atherosclerotic lesions followed by macrophage infiltration as shown by real-time PCR, gel-based and in situ zymography and immunohistochemistry. Expression of other investigated MMPs did not increase during disease progression. However, the mRNA expression of MMP-8 and MMP-13 was down-regulated, which could explain the relatively high amount of collagen observed in the vessels in this model. In conclusion, low proteolytic expression at early stages of atherogenesis and a limited repertoire of proteolytic enzymes were associated with the progression of atherosclerosis in Ldlr(-/-)Apob(100/100) mice. The study suggests that MMP-2 and MMP-9 are the main proteases involved in atherogenesis in this mouse model.

Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory process involving the activity of several cytokines and growth factors. Platelet-derived growth factor-A (PDGF-A) and PDGF-B are important mitogens and chemoattractants for monocytes as well as smooth muscle cells. We sought to identify the role of PDGF-C and PDGF-D, two new members of the PDGF family, in monocyte migration and differentiation. We also assessed their effects in regulating matrix metalloproteinase-2 (MMP-2) and MMP-9, which are important for cell migration. METHODS AND RESULTS: PDGF-C and PDGF-D were expressed in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic plaques, as shown by immunohistochemical analysis. PDGF-C and PDGF-D mRNA and protein expression was induced after differentiation of THP-1 monocytes to macrophages, and both PDGF-C and PDGF-D induced MMP-9 mRNA expression in a concentration-dependent manner. Treatment of cells with PDGF-C or PDGF-D enhanced the secretion of MMP-2 and MMP-9 in a cell-dependent manner. In a migration assay using a Boyden chamber with 8 microm pore size, PDGF-C and PDGF-D attracted THP-1 monocytes in a concentration-dependent manner. CONCLUSIONS: Our data suggest that PDGF-C and PDGF-D, like PDGF-A and PDGF-B, play important roles in atherosclerosis by stimulating MMP activity and influencing monocyte migration.

VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

ADAMTS-4 and -8 are inflammatory regulated enzymes expressed in macrophage-rich areas of human atherosclerotic plaques.

OBJECTIVES: Remodeling of extracellular matrix (ECM) plays an important role in inflammatory disorders such as atherosclerosis. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is a recently described family of proteinases that is able to degrade the ECM proteins aggrecan and versican expressed in blood vessels. The purpose of the present study was to analyze the expression and regulation of several ADAMTSs before and after macrophage differentiation and after stimulation with IFN-gamma, IL-1beta and TNF-alpha. ADAMTS expression was also examined during atherosclerosis development in mice and in human atherosclerotic plaques. METHODS AND RESULTS: Real time RTPCR showed that, of the nine different ADAMTS members examined, only ADAMTS-4 and -8 were induced during monocyte to macrophage differentiation, which was also seen at protein level. Macrophage expression of ADAMTS-4, -7, -8 and -9 mRNA were enhanced upon stimulation with IFN-gamma or TNF-alpha. Furthermore, immunohistochemical analyses revealed that ADAMTS-4 and -8 were expressed in macrophage rich areas of human atherosclerotic carotid plaques and coronary unstable plaques. In addition, ADAMTS-4 expression was upregulated during the development of atherosclerosis in LDLR(-/-)ApoB(100/100) mice. Whereas ADAMTS-4 expression was low in non-atherosclerotic aortas, it was significantly higher in aortas from 30-40-week old atherosclerotic animals. CONCLUSION: The present study suggests that ADAMTS-4 and -8 are inflammatory regulated enzymes expressed in macrophage-rich areas of atherosclerotic plaques. This is the first study associating ADAMTS-4 and -8 expression with atherosclerosis. However, further experiments are required to understand the physiological and pathological functions of ADAMTS in the vascular wall, and tools to measure ADAMTS activity need to be developed.

Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms.

It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture. The growth of the AAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase-9 (MMP-9) and inhibit its degradation, thereby preserving enzymatic activity. Biopsies were obtained from thrombus-free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography, Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself. Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall. Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions. The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

Induction of neutrophil gelatinase-associated lipocalin in vascular injury via activation of nuclear factor-kappaB.

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.

Allele-specific MMP-3 transcription under in vivo conditions.

A common matrix metalloproteinases-3 (MMP-3) -1612 5A/6A promoter polymorphism is associated with risk for cardiovascular disease, rheumatoid arthritis, and other diseases. Here we used the haplotype chromatin immunoprecipitation method to study allele-specific MMP-3 expression under in vivo conditions in heterozygous THP-1 cells. Pyrosequencing was used to analyse the ratio of 5A-allele to 6A-allele after chromatin immunoprecipitation using an antibody against phosphorylated active RNA polymerase II. There was no allele-specific difference in transcriptional activity during basal conditions, i.e., in unstimulated monocytic THP-1 cells. However, after stimulation of MMP-3 expression by monocyte differentiation or incubation with IL-1beta, the haplotype containing the 5A-allele was associated with higher transcriptional activity compared with the 6A-containing haplotype. Electromobility shift assay demonstrated increased binding of nuclear proteins to the 5A-allele after monocyte differentiation. In conclusion, the common MMP-3 5A/6A promoter polymorphism appears to be functional only during specific environmental conditions involving inflammation.

Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) modulates the activity of matrix metalloproteinase (MMP) 9, an important mediator of vascular remodeling and plaque instability in atherosclerosis. This study aimed to analyze the expression of NGAL in atherosclerotic plaques and myocardial infarction (MI). METHODS AND RESULTS: Atherosclerotic apolipoprotein E (apoE)(-/-) x low-density lipoprotein receptor (LDLR)(-/-) and C57BL/6J control mice were exposed to brief hypoxic stress (10 minutes of 10% oxygen). Expression of the mouse NGAL homolog (24p3) and MMP-9 was analyzed 48 hours later by quantitative RT-PCR, immunohistochemistry, and zymography. Hypoxic stress increased NGAL/24p3 mRNA in the cardiac vasculature. NGAL/24p3 was also increased in atherosclerotic plaques of apolipoprotein E(-/-) x LDLR(-/-) mice compared with C57BL/6J mice. Mice developing MI exhibited the highest plaque mRNA expression of NGAL/24p3 and MMP-9. Zymography revealed strong proteolytic activity in areas rich in 24p3 and MMP-9 protein. Immunohistochemistry performed on human carotid endarterectomy specimens and control tissue from the internal mammary artery showed colocalization of MMP-9 and NGAL with macrophages in the atherosclerotic plaques. CONCLUSIONS: NGAL/24p3 is increased in atherosclerotic plaques and MI. Colocalization with MMP-9 in areas with high-proteolytic activity suggests a role for NGAL/24p3 in modulating the MMP-9-mediated remodeling of plaques and infarcted hearts.

Difference in matrix-degrading protease expression and activity between thrombus-free and thrombus-covered wall of abdominal aortic aneurysm.

OBJECTIVE: It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA rupture. Here, we examined the possibility that the intraluminal thrombus influences expression and activity of matrix-degrading proteases in the AAA wall. METHODS AND RESULTS: Twenty patients undergoing elective repair of AAAs were included. From each patient, specimens from both thrombus-covered and thrombus-free wall were taken for analysis. Gene arrays and quantitative real-time polymerase chain reaction showed that matrix metalloproteinase (MMP)-1, -7, -9, and -12 expressions were upregulated in the thrombus-free wall compared with the thrombus-covered wall. Immunohistochemistry confirmed the differential expression of MMP-9 but also localized MMP-9 to the interface between the thrombus and the underlying vessel wall. MMP-9 expression was colocalized with the presence of macrophages. Similar expression patterns were observed for urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-1. Gelatinase activity was detected in the same regions as MMP-9 protein expression, ie, within the thrombus-free wall and in the interface between the thrombus and the underlying wall. CONCLUSIONS: The present work demonstrates that protease expression and activity differs within the aneurysm wall. The source and activity of the proteases responsible for the degradation of the thrombus-covered wall need to be further determined.

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells.

The majority of human melanomas harbor activating mutations in either the BRAF or NRAS gene. To date, the role of oncogenic NRAS in melanoma remains poorly defined and no current therapies are directed at specifically suppressing oncogenic NRAS in human melanoma tumors. The aim of our study, therefore, was to investigate the effects of suppressing oncogenic NRAS in human melanoma cell lines in vitro. Using both small interfering RNA- and plasmid based-RNA interference techniques, oncogenic NRAS was specifically suppressed in 2 human melanoma cell lines, 224 and BL, which harbor a codon 61 CAA (glutamine) to CGA (arginine) NRAS mutation. Suppression of oncogenic NRAS in these cell lines resulted in increased apoptosis. Furthermore, in 224 cells we demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and reduced expression of NF-kappaB and cyclin D1 in the N-Ras signaling pathway. In contrast, RNA interference directed at wild-type (WT) NRAS had no significant effect on apoptosis of 224 cells or 2 human melanoma cell lines (A375 and 397) containing WT NRAS but a codon 600 GTG (valine) to GAG (glutamate) mutation in BRAF. These data suggest that oncogenic NRAS is important for avoidance of apoptosis in melanomas that harbor the codon 61 NRAS mutation and emphasizes oncogenic NRAS as a therapeutic target in patients with tumors that harbor this mutation.