Suppresd Soluble Fms-Like Tyrosine Kina-1 Production Aggravates Atherosclerosis in CKD : Evaluation by Circulating sFlt-1 Levels after Low-Do Heparin Injection
Online supplement
Masaru Matsui, MD1*, Y ukiji T akeda, MD1*, Shiro Uemura, MD1**, T akaki Matsumoto, MD1, Ayako Seno, MD1, Kenji Onoue, MD1, Hideo T sushima, MD1, Katsuhiko Morimoto, MD1, T sunenari Soeda, MD1, Satoshi Okayama, MD1, Satoshi Somekawa, MD1,2, Ken-ichi Samejima, MD1, Hiroyuki Kawata, MD1, Rika Kawakami, MD1, Kimihiko Nakatani, MD1, Masayuki Iwano1***, MD Y oshihiko Saito, MD1,2
*The first 2 authors contributed equally to this work.
黄瓜的功效***Prent affiliation is Department of Nephrology, Fukui University
1First Department of Internal Medicine, Nara Medical University
2Department of Regulatory Medicine for Blood Pressure
Contents
1. Supplemental Methods
2. Supplementary Figure legends
3. Reference
**A ddress for correspondence
Shiro Uemura, MD, PhD申请微信号注册
First Department of Internal Medicine, Nara Medical University,遣悲怀其一
840 Shijo-cho, Kashihara, Nara, 634-8522 Japan,
Phone +81-744-22-3051 Ex.3411; Fax +81-744-22-9726;
E-mail:********************.jp
Supplemental Methods
Clinical definition
We conducted patient interviews and performed laboratory tests to evaluate the following patient background characteristics: age, x, body weight, body mass index, hypertension, diabetes, dyslipidemia, and past history of coronary artery dia (CAD). Hypertension was defined as systolic bl ood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or current treatment wi th oral antihypertensive drugs. Diabetes was defined as fasting gluco ≥ 126 mg/dl or current treatment with oral hypog lycemic medications or insulin. Dyslipidemia was defined as low-density lipoprotein (LDL) cholesterol ≥ 140 mg/dl or current treatment with lipid-lowering medications. Previous CAD was defined by a history of myocardial infarction, angina pectoris, or coronary artery bypass grafting surgery. Patients were diagnod with proteinuria if dipstick test results were "1+" or greater.
Estimated GFR (eGFR) was calculated using the Modification of Diet in Renal Dia equation revid for Japan: eGFR (ml/min/1.73 m2) = 194 ×(rum creatinine)−1.094 × age−0.287 (if female, ×0.739).1CKD stage was categorized according to a modified National Kidney Foundation classification as follows: Control, including Stages 1 and 2 (eGFR ≥ 60 ml/min/1.73 m2); Stage 3a (eGFR 45–59 ml/min/1.73 m2); Stage 3b (eGFR 30–44 ml/min/1.73 m2); Stage 4 (eGFR 15–29 ml/min/1.73 m2); Stage 5 (eGFR < 15 ml/min/1.73 m2); and Stage 5D (hemodialysis patients). Contr
ol subjects consisted of patients visiting our department to undergo cardiac catheterization as well as healthy volunteers.
Cardiovascular events were defined as the composite of the following individual events occurring during the study period: fatal or non-fatal newly developed coronary artery dia, sudden cardiac death, peripheral arterial dia, congestive heart
failure requiring hospitalization, cerebrovascular dia, and aortic dia including rupture and disction of aortic aneurysm.
Generation of sFlt-1−/− and sFlt-1−/− A poE−/−mice
sFlt-1 knockout mice were generated by the inrtion of a targeting vector into the sFlt-1 genomic locus. The constructed targeting vector was introduced into embryonic stem (ES) cells on a C57BL/6 background for homologous recombination (Supplementary Figure 2A). Recombined ES cell clones were identified by PCR, and a successful single inrtion was confirmed by the Southern blot analysis (Supplementary Figure 2B,C). Blastocysts containing ES cells with the floxed allele were implanted and chimeras were bred to obtain heterozygous mice that carry the targeted sFlt-1 locus in their germ line. The floxed PGK-neo castte was removed by breeding with a CAG-Cre tra
nsgenic line. All breeding was done with mice under the C57BL/6 genetic background. Frequency of genotypes obtained from sFlt-1 Het–Het mating was shown in Supplementary Figure 2D. We crosd sFlt-1−/−mice with ApoE−/−mice to obtain sFlt-1−/− ApoE−/− mice.
RT–PCR
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Quantitative real-time polymera chain reaction (RT-PCR) was performed using commercially available or homemade primers and probes for the studied genes. mRNA was extracted from various frozen organ specimens, and cDNA was synthesized according to the standard protocol.
反胃是什么原因Expression levels of Flt-1 mRNA were measured using SYBR green real-time polymera chain reaction with gene-specific primers as follows:
forward : 5´-CCCTGCAACA TTCAGGCACC-3´
rever : 5´-GGCTCGGGGACACCA TTAGC-3´
The combination of forward and rever was designated to detect Flt-1 in human. The levels of genes related to angiogenesis (PlGF, VEGF, Angiopoietin-2), oxidative stress (HO-1, a component of nonphagocyte-type NAHPD oxida: NOX-2), and endothelial damage (Selectin, ICAM-1, VCAM-1)
were detected using T aqman Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Expression levels were normalized by u of glyceraldehyde-3-phosphate dehydrogena as an endogenous control.
Expression of Flt-1 and sFlt-1 mRNA in vascular tissues
HUVECs (Human Umbilical V ein Endothelial Cells), CASMC (Normal Human Coronary Artery Smooth Muscle Cells) were purchad (LONZA; Allendale, NJ, USA) for the analysis of Flt-1 and sFlt-1 mRNA expressions. Monocytes were isolated from human blood taken from volunteer donors by Ficoll-Paque density gradient (Lymphocyte Separation Medium, MP Biomedicals; Irvine, CA, USA).
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Macrophage preparation
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T o harvest peritoneal macrophages, the peritoneal cavity was washed with 5 ml of RPMI 10% FBS.2The pooled cells were then eded in RPMI 10% FBS in 6-well
plates. After 6 hour of incubation at 37 °C in a moist atmosphere of 5% CO2 and 95% air, non-adhering cells on each plate were removed by rinsing with phosphate-buffered saline. The attached macrophages were grown in RPMI 10% for 12 hour, and then harvested for mRNA analysis.
Immunohistochemistry
Paraffin-embedded aortic roots were stained with antibodies against F4/80 (MCA497GA, 1:200; Serotec, Oxford, UK), and subquently incubated with biotinylated condary antibody (Mou anti-Rat IgG: BD Pharmingen, San Diego, CA, USA), followed by amplification with the signal amplification system (CSA System, Dako; Carpinteria, CA, USA).
dnf如何双开Isolated tissues of lung, heart, and muscle were embedded in paraffin, and were ctioned at the thickness of 6-µm with a cryostat as described previously.3Tissues were stained with antibodies against αSMA(1:200; Sigma-Aldrich, Saint Louis, MO, USA ), and CD31 (1:200; BD Pharmingen, San Diego, CA ,USA). V esl density was determined by counting the number of CD31-stained vesls and αSMA-stained vesls per high-power field (magnification, х200) in heart, lung, and muscle, respectively.
Supplementary information is available at Kidney International
