Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells.

BACKGROUND: We evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient. METHODS: EPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban. RESULTS: Rivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair. CONCLUSIONS: Rivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

Mobilization of progenitor cells and vessel healing after implantation of SYNERGY in acute coronary syndrome.

This study was aimed to compare the vascular healing process of a SYNERGY stent with that of a PROMUS PREMIER stent in patients with acute coronary syndrome (ACS). In 71 patients with ACS, undergoing coronary stent implantation using the SYNERGY stent (n = 52) or PROMUS PREMIER stent (n = 19), we measured circulating CD34+/CD133+/CD45null cells and CD34+/KDR+ cells and observed vascular healing at the stented sites using optical coherence tomography (OCT) and coronary angioscopy. On the day 7, circulating CD34+/CD133+/CD45null cells increased in SYNERGY group (P < 0.0001), while it did not change in PROMUS group. The CD34+/KDR+ cells also increased in SYNERGY group (P < 0.0001) but less significantly in the PROMUS group (P < 0.05). The OCT-based neointimal thickness (P < 0.0005) and neointimal coverage rate (P < 0.05) at 12 months were greater in SYNERGY group, compared with PROMUS group. The coronary angioscopy-based neointimal coverage grade at 12 months was also greater in SYNERGY group (P < 0.001). In overall patients, the change in CD34+/KDR+ cells on the day 7 correlated with the OCT-based neointimal thickness at 12 months (R = 0.288, P < 0.05). SYNERGY stent seems to have potential advantages over PROMUS PREMIER stent for ACS patients in terms of vascular healing process at the stented sites.

Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model.

The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient's age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term "wound repair priming." Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat in situ, and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.

Mobilization of progenitor cells and assessment of vessel healing after second generation drug-eluting stenting by optical coherence tomography.

BACKGROUND: Bone marrow-derived progenitor cells likely contribute to both endothelial- and smooth muscle cell-dependent healing responses in stent-injured vessel sites. This study aimed to assess mobilization of progenitor cells and vessel healing after zotarolimus-eluting (ZES) and everolimus-eluting (EES) stents. METHODS AND RESULTS: In 63 patients undergoing coronary stent implantation, we measured circulating CD34 + CD133 + CD45low cells and serum levels of biomarkers relevant to stem cell mobilization. In 31 patients of them, we assessed vessel healing within the stented segment using optical coherence tomography (OCT) imaging. The CD34 + CD133 + CD45low cells increased 68 ± 59% 7 days after bare metal stent (BMS), 10 ± 53% after ZES (P < 0.01 vs BMS), 3 ± 49% after EES (P < 0.001 vs BMS), compared with baseline. Percent change in CD34 + CD133 + CD45low cells was positively correlated with that in stromal cell-derived factor (SDF)-1α (R = 0.29, P = 0.034). Percentage of uncovered struts was higher in the EES group (14.4 ± 17.3%), compared with the BMS (0.7 ± 1.3, P < 0.01) and ZES (0.4 ± 0.5, P < 0.01) groups. The change in CD34 + CD133 + CD45low cells showed positive correlation with OCT-quantified mean neointimal area (R = 0.48, P < 0.01). Finally, circulating mononuclear cells obtained from 5 healthy volunteers were isolated to determine the effect of sirolimus, zotarolimus and everolimus on vascular cell differentiation. The differentiation of mononuclear cells into endothelial-like cells was dose-dependently suppressed by sirolimus, zotarolimus, and everolimus. CONCLUSIONS: Mobilization of progenitor cells was suppressed, and differentiation of mononuclear cells into endothelial-like cells was inhibited, in association with increased number of uncovered stent struts, even after second generation drug-eluting stenting. These data suggest that new approaches are necessary to enhance stent healing.

Spread of X-chromosome inactivation into chromosome 15 is associated with Prader-Willi syndrome phenotype in a boy with a t(X;15)(p21.1;q11.2) translocation.

X-chromosome inactivation (XCI) is an essential mechanism in females that compensates for the genome imbalance between females and males. It is known that XCI can spread into an autosome of patients with X;autosome translocations. The subject was a 5-year-old boy with Prader-Willi syndrome (PWS)-like features including hypotonia, hypo-genitalism, hypo-pigmentation, and developmental delay. G-banding, fluorescent in situ hybridization, BrdU-incorporated replication, human androgen receptor gene locus assay, SNP microarrays, ChIP-on-chip assay, bisulfite sequencing, and real-time RT-PCR were performed. Cytogenetic analyses revealed that the karyotype was 46,XY,der(X)t(X;15)(p21.1;q11.2),-15. In the derivative chromosome, the X and half of the chromosome 15 segments showed late replication. The X segment was maternal, and the chromosome 15 region was paternal, indicating its post-zygotic origin. The two chromosome 15s had a biparental origin. The DNA methylation level was relatively high in the region proximal from the breakpoint, and the level decreased toward the middle of the chromosome 15 region; however, scattered areas of hypermethylation were found in the distal region. The promoter regions of the imprinted SNRPN and the non-imprinted OCA2 genes were completely and half methylated, respectively. However, no methylation was found in the adjacent imprinted gene UBE3A, which contained a lower density of LINE1 repeats. Our findings suggest that XCI spread into the paternal chromosome 15 led to the aberrant hypermethylation of SNRPN and OCA2 and their decreased expression, which contributes to the PWS-like features and hypo-pigmentation of the patient. To our knowledge, this is the first chromosome-wide methylation study in which the DNA methylation level is demonstrated in an autosome subject to XCI.

Cardioprotective effects of low-dose combination therapy with a statin and an angiotensin receptor blocker in a rat myocardial infarction model.

PURPOSE: Statins attenuate angiotensin II-induced myocyte hypertrophy and this might increase the cardioprotective effects of renin-angiotensin system inhibition in the ischemic heart. In this study, we investigated the cardioprotective effects of combination therapy with low-dose simvastatin and low-dose losartan using a rat myocardial infarction model. METHODS: Myocardial infarction was created in rats by left anterior descending artery ligation, and the animals were randomly allocated to one of four groups: control (n=8), losartan 3 mg/kg/day (n=8), simvastatin 2 mg/kg/day (n=8), and losartan 3 mg/kg/day plus simvastatin 2 mg/kg/day (n=8). Each treatment was started on the day of coronary ligation, and hemodynamics, myocardial blood flow, and infarct size were measured after 28 days. RESULTS: Blood pressure, heart rate, and left ventricular systolic and end-diastolic pressures were not significantly different comparing the control group with the 3 other treatment groups. The peak positive first derivative of left ventricular pressure (peak LV dP/dt) was equivalent comparing the control group with the losartan and simvastatin groups. However, the peak LV dP/dt was greater in the losartan plus simvastatin group than in the control group (p<0.05). Myocardial blood flow, left ventricular weight, and infarct size were not significantly altered by the 3 treatments. CONCLUSIONS: Treatment with 3 mg/kg/day losartan plus 2 mg/kg/day simvastatin but not losartan or simvastatin alone improved left ventricular systolic function in a rat myocardial infarction model. The result suggests that statins given in combination with angiotensin receptor blockers might have beneficial cardioprotective effects, even at low-doses for each agent.

Protective effect of n-3 polyunsaturated fatty acid on primary culture of rat hepatocytes.

BACKGROUND AND AIM: Recently, we reported on the beneficial clinical effects of eicosapentaenoic acid (EPA) in patients with primary biliary cirrhosis (PBC) who were unresponsive to ursodeoxycholic acid (UDCA). In this study we examined the effect of EPA on rat hepatocytes in primary culture. METHODS: Hepatocytes were isolated from rat liver by perfusion of collagenase and cultured with or without EPA. Cell damage induced by chenodeoxycholic acid (CDCA) was assessed by WST-8 assay and lactate dehydrogenase (LDH) release. PGE(2) and LTB(4) concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA). cDNA was made from total RNA that was extracted from hepatocytes, and TaqMan polymerase chain reaction (PCR) was performed to assess the expression of CuZn and Mn superoxide dismutase (SOD) mRNA. RESULTS: When rat hepatocytes were cultured in the presence of EPA, the damage caused by CDCA was significantly decreased compared with cells cultured without EPA. Cytotoxicity significantly decreased in the presence of EPA. Furthermore, SOD mRNA expression was increased by adding EPA. These findings indicated that EPA protects cells by scavenging superoxide radicals ((*)O(2-)) mediated by SOD production. CONCLUSION: EPA has a direct protective effect on rat hepatocytes, which is in agreement with the clinical efficacy of EPA in PBC patients.

Mobilization of CD34-positive bone marrow-derived cells after coronary stent implantation: impact on restenosis.

BACKGROUND: Recently, accumulating evidence has indicated that bone marrow-derived stem cells are capable of differentiating into vascular cells. It has been hypothesized that the inflammatory response after vascular injury triggers the mobilization of endothelial and smooth muscle progenitor cells from bone marrow. METHODS AND RESULTS: We measured circulating CD34-positive mononuclear cells, activation of integrin Mac-1 on the surface of neutrophils, and plasma granulocyte-colony stimulating factor levels in 40 patients undergoing coronary stenting. After bare-metal stenting, CD34-positive cells increased, reaching a maximum on day 7 after stenting. The maximum change compared with baseline before stenting was more striking in patients with restenosis than without restenosis (332+/-108% versus 148+/-49%; P<0.05). In contrast, CD34-positive cells decreased after sirolimus-eluting stenting (72+/-21% on day 7). The change in CD34-positive cells on day 7 relative to baseline was closely correlated with that in activated Mac-1 at 48 hours (R=0.52, P<0.01) and that in granulocyte-colony stimulating factor levels at 24 hours (R=0.42, P<0.05). Cell culture assay on day 7 showed that mononuclear cells differentiated into CD31-positive endothelium-like cells after bare-metal stenting. In patients with restenosis, mononuclear cells differentiating into alpha-smooth muscle actin-positive smooth muscle-like cells also were observed. Implantation of sirolimus-eluting stents suppressed both types of differentiation. CONCLUSIONS: Stent implantation may induce differentiation of bone marrow cells into endothelial or smooth muscle cells. Endothelial cells may participate in reendothelialization, a protective reaction against vascular injury, whereas smooth muscle cells may participate in neointimal thickening and restenosis. Sirolimus-eluting stents appear to inhibit the mobilization and differentiation of bone marrow cells.

Effects of histologic type on levels of macrophage colony-stimulating factor in liquid contents of benign ovarian tumors.

BACKGROUND: Normal ovarian tissue is rich in cytokines. Cytokines are important in the physiology of ovarian function. Most of the same cytokines that are found in normal ovarian tissue are also found in association with benign and malignant tumors in contrast to their functions in normal tissues. Thus, we measured macrophage colony-stimulating factor (M-CSF) levels in the liquid contents of benign ovarian tumors--serous cystadenoma, mucinous cystadenoma, and mature cystic teratoma--and investigated whether M-CSF levels were associated with the histologic type of the ovarian tumors. METHODS: We enrolled 65 patients, 52 with benign ovarian tumor and 13 in the early postmenopausal period with symptoms of a menopausal disorder. Among the 52 patients with benign ovarian tumor, 16 had serous cystadenoma, 21 had mucinous cystadenoma, and 15 had mature cystic teratoma. Immediately after surgery, the liquid content was drawn from the ovarian tumor, then centrifuged, and the separated supernatant was stored at -30 degrees C. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay method with use of three antibodies. RESULTS: The level of M-CSF was 12,513 U/mL (median) (range, 0-169,000 U/mL) in serous cystadenoma, 915 U/mL (0-82,500 U/mL) in mucinous cystadenoma, and 149 U/mL (0-6,230 U/mL) in mature cystic teratoma. The M-CSF levels increased significantly from mature cystic teratoma to mucinous cystadenoma to serous cystadenoma. The serum M-CSF levels were 308 to 499 U/mL in patients with benign ovarian tumor. The M-CSF levels did not differ significantly among the three groups. The serum M-CSF levels were 162 U/mL (0-473 U/mL) in menopausal patients. CONCLUSIONS: Elevation of levels of M-CSF varies according to histologic type in benign ovarian tumors. This implies that the antitumor activities of M-CSF for serous cystadenoma, mucinous cystadenoma, and mature cystic teratoma differ by histologic type.

Comparison of changes in circulating platelet-derived microparticles and platelet surface P-selectin expression after coronary stent implantation.

Platelet-derived microparticles (PDMPs) are released from activated platelets and may participate in the inflammatory process in response to vessel wall injury. This study was designed to compare the clinical significance of circulating PDMPs with that of P-selectin on the platelet membrane surface. In 20 patients with stable angina undergoing coronary stent implantation, circulating PDMPs were serially measured by enzyme-linked immunosorbent assay, and P-selectin expression on the surface of platelets was simultaneously analyzed by flow cytometry. PDMPs increased 24-48 h after coronary stenting in the coronary sinus (8.7 +/- 8.9 to 31.8 +/- 19.8 U/ml, P < 0.001) with a maximum at 48 h. In contrast, the mean channel fluorescence intensity for P-selectin increased 15 min after coronary stenting in the coronary sinus (19.5 +/- 5.6 to 25.2 +/- 7.5, P < 0.01) and remained elevated for 48 h; the changes were less striking in peripheral blood. The relative increase in PDMPs was not correlated with the increase in P-selectin expression at 15 min or 24 h after coronary stenting, but was correlated at 48 h (R = 0.48, P < 0.05). Both circulating PDMPs and P-selectin expression were enhanced in association with stent-induced platelet activation; however, the time course of changes in these two platelet activation markers was different. Therefore, the clinical relevance of circulating PDMPs may differ from that of P-selectin expression on the platelet membrane surface.

Granulocyte-macrophage colony-stimulating factor levels in amniotic fluid before the onset of labor and during labor do not differ in normal pregnancies.

PROBLEM: Granulocyte-macrophage colony-stimulating factor (GM-CSF) at the implantation site may regulate invasion and differentiation of placental trophoblast. We evaluated whether GM-CSF levels in amniotic fluid during labor contributing to subsequent delivery differed from those before the onset of labor in normal pregnancies. METHOD OF STUDY: This study enrolled 36 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these pregnancies, 18 were women during labor that led to subsequent term delivery (labors). The other 18 were women without labor underwent cesarean section (controls). These two groups (18 labors and 18 controls) were compared. The average gestational age at entry was 38-39 weeks of gestation. The women's ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the GM-CSF levels were compared between two groups. The GM-CSF level was determined by the enzyme-linked immunosorbent assay method. RESULTS: There was no significant increase in GM-CSF levels in amniotic fluid during labor compared with that before the onset of labor. CONCLUSIONS: The GM-CSF in amniotic fluid may not promote the onset of labor at term and/or term labor contributing to subsequent delivery may not induce the production and secretion of GM-CSF into amniotic cavity.

Tumor necrosis factor-alpha in the placenta is not elevated in pre-eclamptic patients despite its elevation in peripheral blood.

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells at the early and late stages of gestation and promotes the regulation of trophoblast growth and invasion. We evaluated whether TNF-alpha levels in the placenta and blood of pre-eclamptic women differed from those with normal pregnancies. METHOD OF STUDY: The subjects were 39 pregnant women carrying single fetuses (21 normal-pregnant and 18 pre-eclamptic patients). Their average gestational age at entry was 38-39 weeks. Peripheral blood was collected before the onset of labor and separated serum was stored at -20 degrees C. A tissue segment of the placenta was cut and frozen in liquid nitrogen immediately after delivery at -80 degrees C. The frozen placental tissue was added to phosphate-buffered saline. The tissue was fully homogenized and centrifuged. Separated supernatant was stored at -80 degrees C. TNF-alpha levels in separated serum and TNF-alpha and total protein (TP) levels in separated supernatant were measured. The presence of TNF-alpha in the placenta was evaluated by immunohistochemistry in five pre-eclamptic and five normal-pregnant patients. RESULTS: Serum TNF-alpha levels were higher in pre-eclampsia than in normal pregnancies. However, TNF-alpha/TP levels in the placenta did not differ significantly between the two groups. As for TNF-alpha immunostaining of trophoblastic cells in the placenta, it was weak in three and moderate in two of the normal pregnancies, while it was absent in two, weak in one, and moderate in two in the pre-eclampsia group. CONCLUSIONS: We demonstrated no significant increase in TNF-alpha/TP levels in the placenta in pre-eclampsia despite a significant increase in serum TNF-alpha levels. There was no strong immunostaining for TNF-alpha detected by immunohistochemistry in the pre-eclampsia group. These findings suggest that TNF-alpha in the placenta is not a key cytokine to interfere with normal trophoblast invasion into the myometrium in pre-eclampsia, and that sources other than the placenta may contribute to the elevated levels of TNF-alpha found in the circulation of pre-eclamptic patients.

Serum levels of macrophage colony-stimulating factor after cesarean section, vaginal delivery, or laparotomy in normal pregnant women and gynecologic patients.

BACKGROUND: Macrophage colony-stimulating factor (M-CSF) is located in villous cells lining the vessels in the placenta in the third trimester and has been implicated in placental growth and development. Macrophage colony-stimulating factor levels in peripheral blood increased significantly with progression of pregnancy in uncomplicated pregnancies. The serum levels of M-CSF appear to be altered after laparotomy in normal pregnant women and nonpregnant gynecologic patients. Thus, the present study examined changes in serum levels of M-CSF before and after laparotomy and compared these findings between the two groups. Macrophage colony-stimulating factor levels before and after vaginal delivery were also examined. METHODS: Peripheral blood was collected before, 1 day, and 10 days after laparotomy or vaginal delivery from 38 subjects, of whom 14 were normal pregnant women who underwent cesarean section (group 1), 12 were gynecologic patients (group 2), and 12 were normal pregnant women who delivered vaginally (group 3). The M-CSF level was determined by the sandwich ELISA method using three antibodies. RESULTS: In all groups, the serum levels of M-CSF increased significantly 1 day after laparotomy or vaginal delivery, but then decreased significantly after 10 days. The net increase 1 day after laparotomy was significantly lower in group 1 than in group 2. Before and 1 day after laparotomy, the M-CSF levels were significantly higher in group 1 than in group 2, but not 10 days after laparotomy. Changes in M-CSF levels in group 3 were relatively similar to those in group 1. CONCLUSIONS: Serum levels of M-CSF were significantly higher in groups 1 and 3 than in group 2, before laparotomy or vaginal delivery. The M-CSF level increased moderately 1 day after cesarean or vaginal delivery, and it increased remarkably after gynecologic laparotomy. The increases in M-CSF levels postlaparotomy may occur via different mechanisms between groups 1 and 2. Placental removal and termination of pregnancy might contribute to the decrease in M-CSF levels, leading to only a moderate increase in M-CSF levels 1 day after laparotomy in group 1.