Hiroyuki Hao

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue–derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.

Arterial Smooth Muscle Cell Heterogeneity. Implications for Atherosclerosis and Restenosis Development

Abstract—During atheromatous plaque formation or restenosis after angioplasty, smooth muscle cells (SMCs) migrate from the media toward the intima, where they proliferate and undergo phenotypic changes. The mechanisms that regulate these phenomena and, in particular, the phenotypic modulation of intimal SMCs have been the subject of numerous studies and much debate during recent years. One view is that any SMCs present in the media could undergo phenotypic modulation. Alternatively, the seminal observation of Benditt and Benditt that human atheromatous plaques have the features of a monoclonal or an oligoclonal lesion has led to the hypothesis that a predisposed, medial SMC subpopulation could play a crucial role in the production of intimal thickening. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. The concept of SMC heterogeneity is gaining wider acceptance, as shown by the increasing number of publications on this subject. In this review, we discuss the in vitro studies that demonstrate the presence of distinct SMC subpopulations in arteries of various species, including humans. Their specific features and their regulation will be highlighted. Finally, the relevance of an atheroma-prone phenotype to intimal thickening formation will be discussed.

Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome

Background— The endoplasmic reticulum (ER) responds to various stresses by upregulation of ER chaperones, but prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear. Methods and Results— Coronary artery segments were obtained at autopsy from 71 patients, and atherectomy specimens were obtained from 40 patients. Smooth muscle cells and macrophages in the fibrous caps of thin-cap atheroma and ruptured plaques, but not in the fibrous caps of thick-cap atheroma and fibrous plaques, showed a marked increase of ER chaperone expression and apoptotic cells. ER chaperones also showed higher expression in atherectomy specimens from patients with unstable angina pectoris than in specimens from those with stable angina. Expression of 7-ketocholesterol was increased in the fibrous caps of thin-cap atheroma compared with thick-cap atheroma. Treatment of cultured coronary artery smooth muscle cells or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, whereas these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by small interfering RNA decreased ER stress-dependent death of cultured coronary artery smooth muscle cells and THP-1 cells. Conclusions— Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of smooth muscle cells and macrophages may contribute to plaque vulnerability.

Endogenous Angiotensin II Induces Atherosclerotic Plaque Vulnerability and Elicits a Th1 Response in ApoE−/− Mice

Rupture of vulnerable plaques is the main cause of acute cardiovascular events. However, mechanisms responsible for transforming a stable into a vulnerable plaque remain elusive. Angiotensin II, a key regulator of blood pressure homeostasis, has a potential role in atherosclerosis. To study the contribution of angiotensin II in plaque vulnerability, we generated hypertensive hypercholesterolemic ApoE−/− mice with either normal or endogenously increased angiotensin II production (renovascular hypertension models). Hypertensive high angiotensin II ApoE−/− mice developed unstable plaques, whereas in hypertensive normal angiotensin II ApoE−/− mice plaques showed a stable phenotype. Vulnerable plaques from high angiotensin II ApoE−/− mice had thinner fibrous cap (P<0.01), larger lipid core (P<0.01), and increased macrophage content (P<0.01) than even more hypertensive but normal angiotensin II ApoE−/− mice. Moreover, in mice with high angiotensin II, a skewed T helper type 1-like phenotype was observed. Splenocytes from high angiotensin II ApoE−/− mice produced significantly higher amounts of interferon (IFN)-&ggr; than those from ApoE−/− mice with normal angiotensin II; secretion of IL4 and IL10 was not different. In addition, we provide evidence for a direct stimulating effect of angiotensin II on lymphocyte IFN-&ggr; production. These findings suggest a new mechanism in plaque vulnerability demonstrating that angiotensin II, within the context of hypertension and hypercholesterolemia, independently from its hemodynamic effect behaves as a local modulator promoting the induction of vulnerable plaques probably via a T helper switch.

Heterogeneity of Smooth Muscle Cell Populations Cultured From Pig Coronary Artery

Objective — Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. Methods and Results — By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a “hill-and-valley” growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-β2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. Conclusions — Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Phenotypic Modulation of Intima and Media Smooth Muscle Cells in Fatal Cases of Coronary Artery Lesion

Objectives—Characterize the phenotypic features of media and intima coronary artery smooth muscle cells (SMCs) in mildly stenotic plaques, erosions, stable plaques, and in-stent restenosis. Methods and Results—Expression of α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chains (SMMHCs), and smoothelin was investigated by immunohistochemistry followed by morphometric quantification. The cross-sectional area and the expression of cytoskeletal proteins in the media were lower in restenotic lesions and, to a lesser extent, in stable plaques compared with mildly stenotic plaques and erosions. An important expression of α-SMA was detected in the intima of the different lesions; moreover, α-SMA staining was significantly larger in erosions compared with all other conditions. In the same location, a striking decrease of SMMHCs and a disappearance of smoothelin were observed in all situations. Conclusions—Medial atrophy is prevalent in restenotic lesions and stable plaques compared with mildly stenotic plaques and erosions. Intimal SMCs of all situations exhibit a phenotypic profile, suggesting that they have modulated into myofibroblasts (MFs). The high accumulation of α-SMA–positive MFs in erosions compared with stable plaques correlates with the higher appearance of thrombotic complications in this situation.

Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro

We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S- and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with &agr;-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S- and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein. In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes.

Macrophage-Derived Angiopoietin-Like Protein 2 Accelerates Development of Abdominal Aortic Aneurysm

Objective—Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). Methods and Results—Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl2-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. Conclusion—Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.

Role of Endothelial Cell–Derived Angptl2 in Vascular Inflammation Leading to Endothelial Dysfunction and Atherosclerosis Progression

Objective—Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. Approach and Results—Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E–deficient mice (ApoE−/−/Angptl2−/−) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell–derived nitric oxide. Conversely, Angptl2−/− mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-&kgr;B signaling in endothelial cells and increased monocyte/macrophage chemotaxis. Conclusions—Endothelial cell–derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.

Dietary cholesterol withdrawal reduces vascular inflammation and induces coronary plaque stabilization in miniature pigs

OBJECTIVE To study the effect of dietary cholesterol withdrawal on size and composition of LDL-hypercholesterolemia-induced coronary plaques in miniature pigs. METHODS Pigs were on normal chow (control group), on a cholesterol-rich diet for 37 weeks (hypercholesterolemic group) or on a cholesterol-rich diet followed by normal chow for 26 weeks (cholesterol withdrawal group). Endothelial function was assessed with quantitative angiography after intracoronary infusion of acetylcholine, plaque load with intra-coronary ultrasound and plaque composition with image analysis of cross-sections. The effect of porcine serum on coronary smooth muscle cell (SMC) function was studied in vitro. RESULTS Cholesterol-rich diet caused LDL-hypercholesterolemia, increased plasma levels of oxidized LDL (ox-LDL) and C-reactive protein (CRP), and induced endothelial dysfunction and coronary atherosclerosis. Dietary cholesterol withdrawal lowered LDL, ox-LDL and CRP. It restored endothelial function, did not affect plaque size but decreased lipid, ox-LDL and macrophage content. Smooth muscle cells and collagen accumulated within the plaque. Increased smoothelin-to-alpha-smooth muscle actin ratio indicated a more differentiated SMC phenotype. Cholesterol lowering reduced proliferation and apoptosis. In vitro, hypercholesterolemic serum increased SMC apoptosis and decreased SMC migration compared to non-hypercholesterolemic serum. CONCLUSIONS Cholesterol lowering induced coronary plaque stabilization as evidenced by a decrease in lipids, ox-LDL, macrophages, apoptosis and cell proliferation, and an increase in differentiated SMC and collagen. Increased migration and decreased apoptosis of SMC may contribute to the disappearance of the a-cellular core after lipid lowering.