Masanobu Kawakami

Shear stress as an inhibitor of vascular smooth muscle cell proliferation : Role of transforming growth factor-β1 and tissue-type plasminogen activator

We examined whether shear stress can inhibit vascular smooth muscle cell (VSMC) proliferation in vitro directly. Human VSMCs were exposed to fluid flow for 24 hours using a cone-plate apparatus, and their proliferation was inhibited significantly by shear stresses of 1.4 and 2.8 Pa (14 and 28 dyne/cm2), according to the magnitude. Next, we investigated whether transforming growth factor-beta 1 (TGF beta 1), which is known to be an important cytokine that suppresses VSMC proliferation, is the predominant mediator of shear-induced inhibition of VSMC growth. After exposure of VSMCs to shear stress (2.8 Pa) for 24 hours, gene expression of TGF beta 1 and, interestingly, tissue-type plasminogen activator, which converts plasminogen to plasmin, an activator of TGF beta 1, increased twofold and fivefold, respectively. The levels of both latent and active forms of TGF beta 1 in conditioned media of VSMCs exposed to fluid flow increased significantly. An anti-TGF beta 1 antibody reversed shear-induced inhibition of VSMC growth significantly. We concluded that shear stress inhibited VSMC proliferation in vitro and this inhibition was mediated predominantly by TGF beta 1 in an autocrine manner. These data suggest that shear stress plays an important role as an inhibitor of atherogenesis in endothelium-desquamated lesions.

A high frequency of apolipoprotein E4 isoprotein in Japanese patients with late-onset nonfamilial Alzheimer's disease

Phenotypes of apolipoprotein E (apo E) were determined by the iso-electric focusing method in 42 Japanese patients with nonfamilial late-onset Alzheimers disease (AD) and 96 age-matched controls without hyperlipidemia and/or diabetes. There was a striking difference in the distribution of apo E phenotypes between patients with AD and controls (P < 0.0001). Such a difference was mostly attributable to different frequencies of phenotypes E4/3 and E3/3. The apo E4/3 phenotype was detected in 24 (57.1%) of 42 patients with AD, more than six times oftener than in nine (9.4%) of 96 controls. In contrast, apo E3/3, which is the most common apo E phenotype in various ethnic groups, was detected in only 15 (35.7%) patients with AD. These results indicate a strong association between apo E4 isoprotein and Japanese late-onset nonfamilial AD, and that apo E4 is a possible risk factor for the development of this type of AD.

Age-Related Changes in Carotid Artery Flow and Pressure Pulses Possible Implications for Cerebral Microvascular Disease

Background and Purpose— We sought to establish the relation between the pulsatile components of pressure and flow waveforms in the carotid artery and their change with age. Methods— Distention (pressure) and axial flow velocity waveforms were recorded noninvasively and simultaneously from the common carotid artery of 56 healthy subjects aged 20 to 72 years. Results— There was a close relation between the time intervals of pressure and flow waves: from foot to first shoulder or peak, to second shoulder or peak, and to incisura (r=0.97, P<0.0001 for each), which approximated the line of identity. The peak and nadir of flow velocity decreased with age, but late systolic flow augmentation increased substantially (1.6 times in the older group); this can be attributed to earlier wave reflection from the lower body. Pressure augmentation index (PAI) and flow augmentation index (FAI) increased similarly with age (PAI (%)=0.84×age−26.6; FAI (%)=0.75×age+11.9; both P<0.0001). Conclusions— Arterial stiffening with aging increases carotid flow augmentation and can explain the increasing flow fluctuations in cerebral blood vessels. Measurement of carotid FAI may provide a gauge for risk of cerebral microvascular damage, just as PAI provides a gauge for risk of left ventricular hypertrophy and failure.

Cachectin/TNF as well as interleukin-1 induces prostacyclin synthesis in cultured vascular endothelial cells.

Cachectin/tumor necrosis factor (cachectin/TNF) has been shown to be capable of stimulating prostacyclin (PGI2) production by vascular endothelial cells in vitro. The stimulation of PGI2 by cachectin/TNF is comparable to that observed with interleukin-1, the monokine previously suggested to be the principal mediator of this effect. The ability of cachectin/TNF to stimulate PGI2 production suggests that it may play a role in producing depressed blood pressure or shock. If so, it might be possible to prevent such adverse effects with the aid of anti-inflammatory agents.

Recombinant human interleukin 1 alpha and beta stimulate mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.

Tumor necrosis factor type α (cachectin) stimulates mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

Abstract To elucidate the mechanism of tumor necrosis factor alpha (TNF-α)-induced bone resorption, the effects of recombinant human TNF-α on mouse osteoblast-like cells (MC3T3-E1) were studied. TNF-α stimulated MC3T3-E1 cells to produce prostaglandin E2 (PGE2) and macrophage colony stimulating activity (M-CSA) in a dose-dependent manner. TNF decreased alkaline phosphatase (AL-P) activity of MC3T3-E1 cells. These TNF effects were observed at 1 ng/ml (∼6×10−11M). The inhibitory effect on AL-P activity was reversible and the cell growth of MC3T3-E1 cells was only slightly affected by TNF. These findings suggest that both PGE2 and M-CSA stimulated by TNF-α are possibly involved in osteoblast-mediated osteoclastic bone resorption, whereas inhibition of AL-P activity may lead to a decrease in bone formation.

Enhanced expression of platelet-derived growth factor-β receptor by high glucose. Involvement of platelet-derived growth factor in diabetic angiopathy

Coronary heart disease is a major complication of diabetic subjects, and platelet-derived growth factor (PDGF) has been implicated in the development of atherosclerosis. We investigated the effects of high glucose on expression of PDGF-β receptor. In a binding assay with 125I-labeled PDGF-BB homodimer, high concentrations of glucose increased high-affinity binding of PDGF-BB on human monocyte-derived macrophages and rabbit aortic medial smooth muscle cells. Northern blot analysis confirmed the enhanced effect of glucose on expression of PDGF-β receptor mRNA in human monocyte-derived macrophages. The protein kinase C inhibitor, staurosporin, completely suppressed an increase in PDGF-BB binding by high glucose, and high glucose significantly activated protein kinase C. These results indicated that PDGF-β receptor expression was enhanced by high glucose through the activation of protein kinase C. Furthermore, we observed similar effects of high glucose on both PDGF-β receptor expression and protein kinase C activation in rat mesangial cells and human capillary endothelial cells. Our results suggest that stimulation of the PDGF system is significantly involved in the development not only of diabetic atherosclerosis but also of microangiopathy.

ENDOTHELIAL CELL INJURY IN VENULE AND CAPILLARY INDUCED BY CONTRAST ULTRASONOGRAPHY

The aim of the present study was to test the hypothesis that microvascular endothelial cells (EC) are subject to the bioeffects induced by contrast ultrasound (US) because of their proximity to the circulating microbubbles. We examined EC injury in each microvessel section (arteriole, capillary or venule) in rat mesenteries among the following five groups: three controls (sham operation, microbubble injection alone, US exposure with saline injection), and two contrast-US groups (US exposure at a 1-Hz or 30-Hz frame rate with microbubble injection). Propidium iodide (PI), a fluorescent indicator of cell injury, was employed to visualize impaired EC. PI-positive nuclei were equally few among the three controls. Contrast-US increased PI-positive cells in capillaries (1-Hz frame rate, 2.4 +/- 2.2 cells per 0.1-mm vessel length, p = 0.09; 30-Hz frame rate, 4.3 +/- 1.8 cells, p < 0.01) and in venules (1-Hz frame rate, 4.1 +/- 2.5 cells, p < 0.05; 30-Hz frame rate, 13.8 +/- 3.6 cells, p < 0.01) compared with sham operation (0.10 +/- 0.22 cells). The finding indicates that diagnostic contrast US potentially causes EC injury, particularly in venules and capillaries.

Peak C-Reactive Protein Level Predicts Long-Term Outcomes in Type B Acute Aortic Dissection

Acute aortic dissection (AAD) is associated with an inflammatory reaction, as evidenced by elevated inflammatory markers, including C-reactive protein (CRP). The association between the peak CRP level and long-term outcomes in type B AAD has not been systematically investigated. The purpose of this study was to investigate whether the peak CRP level during admission predicts long-term outcomes in type B AAD. We conducted a clinical follow-up study of type B AAD. We divided the study population into 4 groups according to the tertiles of peak CRP levels (T1: 0.60 to 9.37 mg/dL; T2: 9.61 to 14.87 mg/dL; T3: 14.90 to 32.60 mg/dL; and unavailable peak CRP group). Multivariate Cox regression analysis was applied to investigate whether the tertiles of peak CRP predict adverse events even after adjusting for other variables. A total of 232 type B AAD patients were included in this analysis. The median follow-up period was 50 months. CRP reached its peak on day 4.5±1.7. Mean peak CRP values in T1, T2, and T3 were 6.4±2.4, 12.0±1.5, and 19.5±4.0 mg/dL, respectively. There were 65 events (39 deaths and 26 aortic events) during the follow-up. T3 and T2 (versus T1) were strong predictors of adverse events (T3: hazard ratio: 6.02 [95% CI: 2.44 to 14.87], P=0.0001; T2: hazard ratio: 3.25 [95% CI: 1.37 to 7.71], P=0.01) after controlling for all of the confounding factors. In conclusion, peak CRP is a strong predictor for adverse long-term events in patients with type B AAD.

Spermine, a Natural Polyamine, Suppresses LFA-1 Expression on Human Lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3+, CD4+, CD8+, CD19+, CD45RA+, CD45RO+, CD4+CD45RA+, CD4+CD45RO+, CD8+CD45RA+, CD8+CD45RO+). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = −0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.