Yukio Ishikawa

The effects of a myocardial bridge on coronary atherosclerosis and ischaemia

The term myocardial bridge (MB) describes the surprisingly common situation in which part of the left anterior descending coronary artery (LAD), running in epicardial adipose tissue, is covered by a bridge of myocardial tissue. The presence of an MB may influence arterial tissue through the alteration of haemodynamic forces by the myocardial contraction of the bridge itself. Histopathologically and ultrastructurally, any manifestations of atherosclerosis elsewhere in the LAD are suppressed in the intima beneath the MB. By scanning electron microscopy, abrupt changes in endothelial cell morphology indicate that the intima beneath the bridge is protected by haemodynamic factors. Furthermore, the closer the bridge to the left coronary ostium, the greater the extent of proximal intimal thickening. In parallel with this, considering the occurrence of myocardial infarction in cases of proximal MB together with previous reports on relationships between MB and coronary ischaemia, it appears that anatomical characteristics such as the location, length, and thickness of the MB have a bearing on the effects of this abnormality. When the pathologist examines the heart at autopsy, this quite common condition should be borne in mind, in view of its potential but complex relationship to atherosclerosis and ischaemic heart disease.

Expression of ACE and ACE2 in Individuals With Diabetic Kidney Disease and Healthy Controls

BACKGROUND Angiotensin-converting enzyme (ACE) 2 (ACE2) is expressed mainly in the heart and kidney and forms angiotensin-1-7 from angiotensin II. ACE2 might act in a counterregulatory manner to ACE. There is little information about renal ACE and ACE2 expression in human diabetic nephropathy. STUDY DESIGN Cross-sectional study. SETTING & PARTICIPANTS Kidney tissue from 20 patients with type 2 diabetes and overt nephropathy and 20 healthy kidney donors. PREDICTOR Diabetes status. OUTCOMES & MEASUREMENTS Renal expression of ACE and ACE2 assessed by means of immunohistochemistry and in situ hybridization. Correlation between ACE and ACE2 expression and levels of various biochemical parameters. RESULTS Decreased ACE2 and increased ACE expression in both the tubulointerstitium and glomeruli resulted in a significant (P < 0.001) increase in ACE/ACE2 ratio in patients with diabetes with overt nephropathy compared with controls, although ACE messenger RNA in the tubulointerstitium did not significantly increase. ACE/ACE2 ratio correlated positively with values for mean blood pressure, fasting blood glucose, serum creatinine, proteinuria, and hemoglobin A(1c) and inversely with estimated glomerular filtration rate (P < 0.001). LIMITATIONS Inclusion of small number of human renal biopsy specimens with structural distortion of cortical tissue. CONCLUSIONS The high ACE/ACE2 ratio in kidneys of patients with type 2 diabetes with overt nephropathy may contribute to renal injury.

Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells

Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.

The effect of myocardial bridging of the coronary artery on vasoactive agents and atherosclerosis localization

The relationship between alterations in the immunohistochemical expression of three vasoactive agents [endothelial nitric oxide synthase (eNOS), endothelin‐1 (ET‐1), and angiotensin‐converting enzyme (ACE)] and the occurrence human atherosclerosis was investigated in relation to the myocardial bridge (MB) of the left anterior descending coronary artery (LAD), an anatomical site that experiences increased shear stress. Five millimetre cross‐sections of LADs with MB from 22 autopsied cases were taken from the left coronary ostium to the cardiac apex and were immunohistochemically stained with antibodies against eNOS, ET‐1, and ACE. The extent of atherosclerosis in each section was calculated using the atherosclerosis ratio (intimal cross‐sectional area/medial cross‐sectional area) determined by histomorphometry. The results were analysed according to their anatomical location relative to the MB, either proximal, beneath, or distal. The extent of atherosclerosis was significantly lower beneath the MB, compared with proximal and distal segments. The expression of eNOS, ET‐1, and ACE was also significantly lower beneath the MB. The expression of these agents correlated significantly with the extent of atherosclerosis. Because nitric oxide, after its production by eNOS, is believed to be degraded by superoxide radicals, the effect of eNOS expression on atherosclerosis remains controversial. However, the present findings clearly indicate that the expression of ET‐1 and ACE is directly related to the development of human coronary atherosclerosis in vivo through shear stress. Copyright

Histopathologic Determinants of Regional Lymph Node Metastasis in Early Colorectal Cancer

Early colorectal cancer (ECC) is curable by endoscopic local resection; however, 10% of patients with ECC exhibit lymph node (LN) metastasis. In the current study, accurate predictors for LN metastasis in patients with ECC were examined by using immunohistochemistry with the lymphatic endothelial hyaluronan receptor 1 (LYVE‐1) antibody to discriminate between lymphatics and blood vessels.

Immunohistochemical detection of human small lymphatic vessels under normal and pathological conditions using the LYVE-1 antibody.

The spread of tumor cells via lymphatic vessels to the lymph nodes is an important indicator of malignancy. However, previous markers used to identify lymphatic endothelium gave ambiguous results in immunohistochemical analyses with paraffin-embedded tissues. In this study, we attempted to prepare a polyclonal antibody against human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) for detecting lymphatic vessels using immunohistochemistry. The antibody was raised against a region near the transmembrane anchor of LYVE-1 in New Zealand white rabbits. Immunostainings with anti-LYVE-1 and von Willebrand factor antibodies were performed in various normal and pathological tissues. LYVE-1 expression was confined to the endothelial surface of lymphatic vessels but was not found in the endothelium of blood vessels, which were positive for von Willebrand factor. Our LYVE-1 polyclonal antibody was useful for the identification of small lymphatic vessels in normal human tissues. In addition, the immunostaining enabled us to distinguish lymphatic invasion by malignant tumor cells from blood vessel invasion using paraffin-embedded sections. In conclusion, our polyclonal antibody against the transmembrane anchor of the peptide can be used to detect human lymphatic vessels under various conditions.

Basic fibroblast growth factor in an artificial dermis promotes apoptosis and inhibits expression of α‐smooth muscle actin, leading to reduction of wound contraction

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of α‐smooth muscle actin (α‐SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 μg) and/or fibroblasts (1 × 106 cells/cm2) applied to experimentally produced full‐thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast‐seeded collagen sponge (fibroblast‐seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF‐treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased α‐SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in α‐SMA‐positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of α‐SMA‐positive fibroblastic cells and inhibits α‐SMA expression in the treated wound, thus reducing wound contraction.

Microsomal prostaglandin E synthase-1 in both cancer cells and hosts contributes to tumour growth, invasion and metastasis.

mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel™ invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.

Myocardial apoptosis associated with the expression of proinflammatory cytokines during the course of myocardial infarction.

To clarify the role of myocardial apoptosis associated with the expression of proinflammatory cytokines in human myocardial infarction (MI), we have analyzed the expression of apoptosis positive for single-stranded DNA (ss-DNA) antibody, tumor necrosis factor (TNF)-α, and interleukin (IL)-8 in 147 samples of infarcted myocardial tissue from 65 patients. ss-DNA-positive apoptotic nuclei were found mainly in cardiomyocytes in the border zones and granulation tissue cells in the infarct foci. The ss-DNA index (SI) of cardiomyocytes (average 0.13%) peaked at stage II (established myocardial necrosis), the value being significantly higher than at stages III (macrophage infiltration), IV (granulation formation), and V (scar formation) (P<0.05), whereas the SI of granulation tissue (average 0.08%) at stages III, IV, and V showed no significant differences between the three stages. These results suggest that cardiomyocyte apoptosis in the border zone is responsible for cellular loss in the acute stage of MI, whereas granulation tissue apoptosis may not be involved in the process of ventricular remodeling. TNF-α was expressed in cardiomyocytes in the border zones of infarct foci, but no significant positive correlation was found between SI and TNF-α index in cardiomyocytes (r=0.08, P=0.37), suggesting that TNF-α does not serve as a direct trigger of cardiomyocyte apoptosis in vivo. The number of IL-8-positive cells peaked at stage II, and IL-8-myeloperoxidase-double-positive neutrophils were frequently detected, indicating that infiltrating neutrophils are the predominant source of IL-8 in the infarcted myocardium. These results suggest that, in human MI, TNF-α produced by cardiomyocytes does not play a critical role in their apoptosis, and that IL-8 produced by neutrophils is responsible for the subsequent accumulation and activation of neutrophils, thus increasing the degree of myocardial damage.

Changes in the distribution pattern of gelatin-binding protein of 28 kDa (adiponectin) in myocardial remodelling after ischaemic injury.

Aims:  Gelatin‐binding protein of 28 kDa (GBP28) is a collagen‐like plasma protein having a binding capacity with collagens. We investigated GBP28 role on myocardial remodelling as well as the diagnostic significance of GBP28 immunostaining in myocardial infarction.