Akiko Kojima

Vascular Endothelial Growth Factor (VEGF) Expression in Human Coronary Atherosclerotic Lesions Possible Pathophysiological Significance of VEGF in Progression of Atherosclerosis

BACKGROUND Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. METHODS AND RESULTS Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription-polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression. Hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. CONCLUSIONS These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Enhanced expression of angiotensin-converting enzyme is associated with progression of coronary atherosclerosis in humans

Background The clinical usefulness of angiotensin converting enzyme (ACE) inhibitors in preventing the recurrence of myocardial infarction has been investigated in large randomized trials. Results from many studies using animal models have suggested that ACE inhibitors have vasculoprotective effects, which may contribute to the prevention of coronary atherosclerosis. Objective To examine the association between vascular angiotensin generation and the development of coronary atherosclerosis in humans. Methods We used immunocytochemical techniques to examine frozen sections from 44 coronary artery segments from 19 corpses. Results Three segments were sites of plaque rupture in patients who had died from acute myocardial infarction. Other specimens of coronary artery segments were characterized histologically to be normal artery segments with diffuse intimal thickening (n = 6), hypercellular lesions composed of smooth muscle cells with or without infiltration of macrophages (n = 11), atheromatous plaque (n = 12), and fibrosclerotic plaque (n = 12). In normal arteries with diffuse intimal thickening, ACE was expressed in endothelial cells. In those with hypercellular lesions and atheromatous plaques, however, enhanced ACE expression was found in macrophages and smooth muscle cells. In contrast, arteries with fibrosclerotic plaques exhibited little or no ACE expression within the plaque. All three ruptured plaques expressed ACE strongly in macrophages accumulated around the attenuated fibrous cap. Conclusion The strong association of enhanced ACE expression with the histologic characteristics of plaques suggests that ACE in hypercellular lesions, atheromatous plaques, and ruptured plaques contributes greatly to the further progression of atherosclerosis via an increase in vascular angiotensin II formation and inactivation of bradykinin.

Relative localization of angiotensin-converting enzyme, chymase and angiotensin II in human coronary atherosclerotic lesions.

BACKGROUND Studies using cell cultures and animal models have indicated an important role for angiotensin II in atherosclerosis. In humans, at least two major enzymes are involved in the conversion of angiotensin I to angiotensin II: so-called angiotensin-converting enzyme (ACE) and chymase. Enhanced activation of chymase in atherosclerotic tissue homogenates has been reported in animal models, but its contribution to the generation of angiotensin II has not been studied. OBJECTIVE To clarify the localization of chymase and its pathophysiologic role in the formation of angiotensin II, using human coronary arteries. DESIGN AND METHODS Twenty-four coronary artery segments obtained from 14 autopsied patients were characterized histologically into the following categories: normal coronary arteries with diffuse intimal thickening, hypercellular lesions, atheromatous plaques and fibrosclerotic plaques. We compared the cellular localization of chymase, ACE and angiotensin II expression using immunocytochemical techniques. RESULTS Chymase was expressed only in the cytosole of mast cells in all segments. On the basis of the histologic study, the number of chymase-positive cells in the intima of atheromatous plaques was significantly higher than that in normal coronary arteries with diffuse intimal thickening. The expression of angiotensin II in the intima was enhanced in hypercellular lesions and atheromatous plaques. Localization of angiotensin II in the intima was associated with that of ACE. Immunodouble staining did not show colocalization of angiotensin II and chymase. CONCLUSIONS These results suggest an important role for the production of angiotensin II by ACE in the progression of atherosclerosis in human coronary arteries. Enhanced expression of chymase appears not to be involved in angiotensin II production in the intima.

Smooth muscle cell de-differentiation is a fundamental change preceding wound healing after percutaneous transluminal coronary angioplasty in humans

Background.Wound healing at the site of medial injury after percutaneous transluminal coronary angioplasty (PTCA) is dominated by smooth muscle cells. This reaction may also cause restenosis. Division and migration of smooth muscle cells relate closely to their cytoskeletal features, as shown experimentally, but in humans little information is available regarding smooth muscle cell activity in post-angioplasty coronary arteries. Materials and methods.This study is based on eight dilated coronary arteries obtained at autopsy from six patients who died within 4 months of an initially successful PTCA. In each patient, a single PTCA had been performed and the target site was identified, sectioned serially, and studied with conventional and immunohistochemical techniques. Results.All target sites showed laceration extending into the media. Two days after PTCA the site of injury was covered by a fibrin-platelet thrombus. The smooth muscle cells of the pre-existent media, immediately adjacent to the site of injury, showed loss of staining for both muscle actin and smooth muscle cell actin, using the antibodies HHF-35 (an anti-muscle actin marker) and CGA-7 (an anti-smooth muscle cell actin marker), respectively. Five days after PTCA, this area had expanded; a distinct influx of macrophages was apparent. From 12 days onwards, the staining density with HHF-35 in the pre-existent media increased and was almost restored to normal at 20 days, but staining with CGA-7 was retarded until approximately 4 months after PTCA. In the repair tissue, spindle-shaped cells were first seen 5 days after PTCA. These cells stained positive with vimentin but did not stain with either actin marker. Macrophages were present at this stage. At 12 days after PTCA, some spindle-shaped cells stained positive with HHF-35, but all were negative with CGA-7. At 16 days, the staining density with HHF-35 had increased, but CGA-7 was still negative. At 20 days, the maximal staining density with HHF-35 was obtained. The vast majority of spindle-shaped cells also stained positive with CGA-7 4 months after PTCA. Endothelial cells on the luminal surface were first identified 4 months after PTCA. Conclusion.The observations provide support that cytoskeletal changes observed experimentally also play a role in human coronary arteries after PTCA. De-differentiation of smooth muscle cells of the pre-existent media, preceding a noticeable cellular response, appears to be a fundamental process.

Growth inhibitory effect of green tea extract and (-)-epigallocatechin in Ehrlich ascites tumor cells involves a cellular thiol-dependent activation of mitogenic-activated protein kinases.

The effect of green tea extract (GTE) in Ehrlich ascites tumor cells (EATC) was studied with respect to changes in the intracellular kinase system involving mitogen-activated protein kinases (MAPKs) and cellular thiol. We have previously shown a reduction in viability of EATC and tyrosine phosphorylation of 42 and 45 kDa proteins by GTE and its polyphenolic component, Epigallocatechin (EGC) (D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yoshihisa, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem. Biol. Interact. 110 (1998) 159-172). Furthermore, GTE and EGC significantly decreased both cellular non-protein and protein sulfhydryl levels in EATC, but replenishing thiol stores with N-acetylcysteine (NAC) caused a recovery in cell viability, and therefore SH groups were identified as a novel target of green tea cytotoxicity (D.O. Kennedy, M. Matsumoto, A. Kojima, I. Matsui-Yuasa, Cellular thiol status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells, Chem. Biol. Interact. 122 (1999) 59-71). In this study, we have observed the stimulation of three forms of MAPK, namely ERK1/2, JNK/SAPK and p38, by EGC, which were dose and time-dependent. These MAPK stimulations were found to be cellular thiol status-dependent events as NAC reversed these stimulations. Furthermore, inhibition of the p38 MAPK pathway using the p38 inhibitor SB203580 caused a marked dose-dependent reduction in the decrease in cell viability caused by EGC treatment. Inhibiting the Erk1/2 MAPK pathway using the MEK inhibitor PD098059 caused a slight change in the decrease in cell viability by EGC. These may suggest that the cytotoxicity associated with EGC was more associated with the other MAPKs than with ERK1/2. This may be the first study of its kind providing a novel evidence of a role for different forms of MAPKs in the antitumor effect of green tea polyphenols, especially EGC, in Ehrlich ascites tumor cells.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of retinoblastoma protein phosphorylation by (-)-epigallocatechin.

Tea polyphenols have been shown to inhibit tumor cell growth, but there is limited information on their effects on cell signaling and cell cycle control pathways. We have shown the involvement of such mechanisms as activation of mitogenic activated protein kinases, decreases in ornithine decarboxylase activity and in cellular thiol levels, elicitation of mitochondrial cytochrome c release, and activation of caspases by the green tea galloyl polyphenol, epigallocatechin (EGC). In the current study, we sought to determine how EGC alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells. The significant finding here is that EGC caused a dose-dependent accumulation of cells in the G1 phase and a decrease in the phosphorylation of the retinoblastoma (Rb) protein, which was also in a cellular thiol-dependent manner. The involvement of a cellular thiol-dependent modulation in Rb phosphorylation leading to the regulation of tumor cell growth by a green tea polyphenol is a novel observation, to the best of our knowledge.

Growth inhibitory effect of green tea extract in Ehrlich ascites tumor cells involves cytochrome c release and caspase activation

We reported previously that the mechanism by which Green tea extract (GTE) elicited growth-inhibitory effects in Ehrlich ascites tumor cells involved a decrease in ornithine decarboxylase (ODC) activity and in cell viability. Decrease in ODC activity has been associated with apoptotic cell death and we therefore studied changes in cytochrome c release and caspase activation, which characterize apoptosis. GTE caused a dose- and time-dependent increase in caspase-3-like protease activation, preceded by a release of cytochrome c from the mitochondria. Inhibiting the activation of caspase-3 with acetyl-Asp-Glu-Val-Asp-alpha-aldehyde (caspase inhibitor) caused a reversal in the effect on cell viability.

Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells.

Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 microg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 microM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition.

Polyamines and thiols in the cytoprotective effect of L-cysteine and L-methionine on carbon tetrachloride-induced hepatotoxicity

Summary. The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamines on the cytoprotective ability of L-cysteine and L-methionine, the most important components in the sulfur amino acid metabolic pathway, in carbon tetrachloride (CCl4)-induced toxicity in isolated rat hepatocytes was studied. CCl4 induced a LDH release and decreased cellular thiols and polyamines levels but treatment with L-cysteine and L-methionine reversed these decreases. Treating with methylglyoxal bis-(guanylhydrazone), MGBG, an irreversible inhibitor of S-adenosylmethionine decarboxylase, which is a key enzyme in spermidine and spermine biosynthesis, and therefore used to deplete cellular polyamines, prevented the protective effect of L-cysteine and L-methionine, but the addition of exogenous polyamines inhibited the influence of MGBG. These results suggest that the cytoprotective effect of L-cysteine and L-methionine in CCl4-induced toxicity were via maintenance of cellular polyamines, GSH and protein-SH concentrations and prevention of LDH leakage.

Globulin and Albumin-2 Associated with Protein Bodies in Amaranthus cruentus Seeds.

To examine whether albumin-2, a specific protein found only in amaranth seeds so far, is associated with protein bodies, we isolated protein bodies from Amaranthus cruentus seed embryos by rate-zonal centrifugation with a sucrose gradient. Most protein bodies in the final preparation were intact when observed by electron microscopy. Profiles by SDS-PAGE showed that the isolated protein bodies contained globulin and albumin-2.