Masako Mitsumata

Laminar Shear Stress Inhibits Vascular Endothelial Cell Proliferation by Inducing Cyclin-Dependent Kinase Inhibitor p21Sdi1/Cip1/Waf1

Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and human umbilical vein ECs, determined by [(3)H]thymidine incorporation, was inhibited by steady laminar shear stress (5 and 30 dyne/cm(2)). This growth inhibition due to shear stress was associated with suppression of cell transition from the G(1) to S phase of the cell cycle. Therefore, we studied G(1)-phase events to find the molecules responsible for this cell cycle arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21(Sdi1/Cip1/Waf1) protein, but not that of p27(Kip1), increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis through the induction of EC stability disruption.

Clinicopathologic Findings in Polypoidal Choroidal Vasculopathy

PURPOSE To elucidate the pathogenic mechanism of polypoidal choroidal vasculopathy (PCV) based on histopathologic findings. METHODS Specimens obtained by surgical excision of PCV from five eyes of five patients (mean age, 75.6 +/- 3.1 years) were studied histopathologically. Immunohistochemical studies were also performed to identify CD34, vascular endothelial growth factor (VEGF), CD68, alpha-smooth muscle actin (alpha-SMA) and hypoxia-inducible factor (HIF)-1alpha. RESULTS Hyalinization of choroidal vessels and massive exudation of fibrin and blood plasma were observed in all the specimens of PCV lesions. Some blood vessels were located above the RPE in two of the five eyes. Immunohistochemically, CD68-positive cells were detected around the hyalinized vessels. There were no alpha-SMA-positive cells in the vessels of PCV. CD34 staining showed endothelial discontinuity. Vascular endothelial cells within the PCV specimens were negative for VEGF. HIF-1alpha positive inflammatory cells were located in the stroma of specimens. CONCLUSIONS Hyalinization of choroidal vessels, like arteriosclerosis, is characteristic of PCV.

Repetitive Fluctuations in Blood Glucose Enhance Monocyte Adhesion to the Endothelium of Rat Thoracic Aorta

Background—The aim of this study was to elucidate the effect of repetitive fluctuations in blood glucose concentrations on monocyte adhesion to the aortic endothelium. Methods and Results—Nonobese type 2 diabetes, Goto–Kakizaki (GK) rats were fed twice daily to induce repetitive postprandial glucose spikes. Then, we compared the number of monocytes adherent to the endothelium of thoracic aorta in these rats with that in rats fed ad libitum. To suppress the glucose spikes, rats were injected with an inhibitor of sodium–glucose transporter, phloridzin, just before each meal for 12 weeks. GK rats fed twice daily showed significantly lower HbA1c than GK rats fed ad libitum. However, the former group showed markedly higher number of monocytes adherent to the endothelium than the latter, together with increased arterial intimal thickening. Phloridzin significantly reduced the number of adherent monocytes in GK rats fed twice daily. Conclusion—Our data demonstrated that repetitive postprandial fluctuation in glucose concentration evokes monocyte adhesion to endothelial cells that was worse than that induced by stable hyperglycemia in vivo. Suppression of such fluctuations efficiently suppressed monocyte adhesion to the aortic endothelium.

Fluid Shear Stress Induces Lipocalin-Type Prostaglandin D2 Synthase Expression in Vascular Endothelial Cells

Abstract —Ligands for peroxisome proliferator–activated receptor γ, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D 2 (PGD 2 ), the metabolic precursor of 15d-PGJ 2 , we identified PGD 2 from culture medium of endothelial cells. To study how PGD 2 production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD 2 . Endothelial cells expressed the mRNA for the lipocalin-type PGD 2 synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm 2 . The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI 2 synthase and thromboxane A 2 synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD 2 and 15d-PGJ 2 into culture medium. These results demonstrate that shear stress promotes PGD 2 production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator–activated receptor γ ligand is produced in vascular wall in response to blood flow.

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Second nation-wide study of atherosclerosis in infants, children and young adults in Japan

Abstract This paper reports the results of a nation-wide cooperative study of atherosclerosis in young, first generation Japanese with ages ranging from 1 month to 39 years, who were autopsied between 1978 and 1982 in hospitals distributed over the entire archipelago of Japan. Atherosclerotic lesions in 2320 aortas, 1620 coronary arteries and 344 cerebral arteries were classified into fatty streaks, fibrous plaques and complicated lesions and were then quantificated with the point-counting method. Atherosclerosis of aortas, coronary arteries and cerebral arteries, determined by surface involvement (SI) of atherosclerotic lesions and atherosclerotic index (AI), increased with age; the severest were seen in aortas, and then, with decreasing severity, in the coronary and cerebral arteries. Fatty streaks preceded the other lesions and accounted for the largest portion of the lesions in aortas and coronary arteries. Fibrous plaques and complicated lesions developed in the later decades of life. The patients with collagen diseases had a greater severity of aortic atherosclerosis in the 2nd and 3rd decades of life, than those without such disorders. Correlation of antemortem clinical data with SI and Al of each artery were analyzed, using simple correlation analysis and multiple regression analysis. Age, serum cholesterol and blood pressure were significantly and positively correlated with SI and AI of aortas and coronary arteries. Serum cholesterol was more strongly correlated with the extent of fatty streaks than was mean blood pressure and vice versa with that of fibrous plaques. Atherosclerosis of cerebral arteries, however, showed a significant correlation only with the factor of mean blood pressure. Therefore the susceptibility to risk factors varies with the artery in cases of early lesions of atherosclerosis in young Japanese.

Hemodynamic-Force-Induced Difference of Interendothelial Junctional Complexesa

1. The flow divider of the brachiocephalic branching of the rabbit aorta had both high and low shear stress regions, each of which was covered by endothelial cells with low and high permeability respectively, even in normolipidemic intact rabbits. When rabbits were placed on an atherogenic diet, low shear regions were the most vulnerable for lipid deposition, but the high shear regions were spared from deposition. 2. A freeze fracture study revealed that high shear regions both at the brachiocephalic branching and in the surgically coarctated abdominal aorta of rabbits had a more common appearance of zonular type tight junctions. Mean low shear regions had more macular and less zonular type. 3. Cultured porcine aortic endothelial cells exposed to laminar 30 dyn/cm2 shear stress in a flow chamber developed ridges of membranous protein particles at the cell-cell contact. 4. Increases of magnitude and duration of exposure to shear stress enhanced the structure of the protein ridge of the tight junction and immunohistochemical expression of proteins associated with both tight and adherens junctions.

The Expression of a Type II Transmembrane Serine Protease (Seprase) in Human Gastric Carcinoma

Objective: The invasion and metastasis of carcinoma cells require the proteolytic degradation of the extracellular matrix by various cell surface proteases. Among these, seprase is a type II transmembrane serine protease absent in normal tissues and it has been implicated in the invasion of the extracellular matrix by both tumor and stromal cells in human breast carcinoma and melanoma. In the present study, the expression of seprase mRNA, protein and its gelatin-degrading activity in human gastric carcinoma were examined to substantiate the potential role of seprase in gastric carcinoma invasion. Methods: We have examined the seprase expression in human gastric carcinoma (n = 34) by RT-PCR, Western immunoblotting analysis, immunohistochemistry, and gelatin zymography. Results: Immunoblotting analysis using mAb D8 directed against seprase showed that the carcinoma tissues in 26 out of 34 cases of gastric cancer expressed a dimeric form of seprase but their normal counterparts did not. Gelatin zymography confirmed that the isolated seprase exhibited the gelatin-degrading activity and was active. Seprase-expressing carcinoma tissues were more often found in the scirrhous type than in other types of gastric carcinoma. RT-PCR analysis showed that seprase mRNA was present in carcinoma tissues but not in normal tissues. Immunohistochemically, seprase was mainly located in gastric carcinoma cells, weakly in stromal cells and microvessel endothelial cells in the tumor nest, and none in normal cells. Conclusions: Our studies showed the unique expression and localization of seprase in the tumor and stromal cells within human gastric carcinoma but not in normal tissues, suggesting a role of seprase in the invasive and metastatic progression of gastric carcinoma.

Laminar Shear Stress–Induced GRO mRNA and Protein Expression in Endothelial Cells

BACKGROUND The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.

Seprase, a Membrane-Type Serine Protease, Has Different Expression Patterns in Intestinal- and Diffuse-Type Gastric Cancer

Objective: Seprase is an integral membrane serine proteinase with gelatinase activity that may be involved in cancer invasion and metastasis. However, the pathophysiologic significance of its expression in gastric cancer tissue has not been fully elucidated. Methods: Seprase expression and distribution in gastric cancer specimens obtained from 133 patients were examined by immunohistochemistry and immunoblotting. Results: Immunohistochemistry showed that in intestinal-type cancer, which includes well and moderately differentiated adenocarcinoma, seprase immunoreactivity was mainly recognized in the moderately differentiated cells and not in the well differentiated cells. In the diffuse type, which includes poorly differentiated adenocarcinoma and signet ring cell carcinoma, seprase immunoreactivity was seen mainly in cells with poor cell-to-cell junctions. The reactive pattern in the cells was different between moderately differentiated adenocarcinoma and diffuse-type carcinoma. Besides the cytoplasm, the cell membrane also apparently reacted in the former, while only the cytoplasm reacted diffusely in the latter. Seprase immunoreactivity was also recognized in endothelial cells and stromal cells especially adjacent to tumor nests. The immunoreactivity of the stromal cells was more abundant in the intestinal type than in the diffuse type, and these stromal expressions of seprase in the intestinal type correlated with the liver (13/13 = 100% of cases with metastases) or lymph node metastases (33/34 = 97% of cases with metastases). Immunoblotting showed that the levels of seprase protein were higher in intestinal-type cancer than in diffuse-type cancer. Conclusion: These results suggested that there is a difference in seprase expression between intestinal- and diffuse-type gastric cancer; this difference may reflect distinct biological features of these types of cancer.