Yasutomo Fukunaga

Inhibition of Rho-Associated Kinase Results in Suppression of Neointimal Formation of Balloon-Injured Arteries

BACKGROUND Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. METHODS AND RESULTS Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg. kg(-1). day(-1)) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22+/-0.02) compared with vehicle-treated rats (intima/media ratio, 0.92+/-0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03+/-0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27(kip1) in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. CONCLUSIONS We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension.

Natriuretic Peptides/cGMP/cGMP-dependent Protein Kinase Cascades Promote Muscle Mitochondrial Biogenesis and Prevent Obesity.

OBJECTIVE Natriuretic peptides (NPs) have been characterized as vascular hormones that regulate vascular tone via guanylyl cyclase (GC), cyclic GMP (cGMP), and cGMP-dependent protein kinase (cGK). Recent clinical studies have shown that plasma NP levels were lower in subjects with the metabolic syndrome. The present study was conducted to elucidate the roles for NP/cGK cascades in energy metabolism. RESEARCH DESIGN AND METHODS We used three types of genetically engineered mice: brain NP (BNP) transgenic (BNP-Tg), cGK-Tg, and guanylyl cyclase-A (GCA) heterozygous knockout (GCA+/−) mice and analyzed the metabolic consequences of chronic activation of NP/cGK cascades in vivo. We also examined the effect of NPs in cultured myocytes. RESULTS BNP-Tg mice fed on high-fat diet were protected against diet-induced obesity and insulin resistance, and cGK-Tg mice had reduced body weight even on standard diet; surprisingly, giant mitochondria were densely packed in the skeletal muscle. Both mice showed an increase in muscle mitochondrial content and fat oxidation through upregulation of peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC)-1α and PPARδ. The functional NP receptors, GCA and guanylyl cyclase-B, were downregulated by feeding a high-fat diet, while GCA+/− mice showed increases in body weight and glucose intolerance when fed a high-fat diet. NPs directly increased the expression of PGC-1α and PPARδ and mitochondrial content in cultured myocytes. CONCLUSIONS The findings together suggest that NP/cGK cascades can promote muscle mitochondrial biogenesis and fat oxidation, as to prevent obesity and glucose intolerance. The vascular hormone, NP, would contribute to coordinated regulation of oxygen supply and consumption.

Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet

In order to elucidate energy balance in the skeletal muscle, we cloned cDNA of a homologue of uncoupling protein (UCP) from rat skeletal muscle. We also cloned rat UCP‐2 cDNA from rat brown adipose tissue (BAT). The UCP cloned from rat skeletal muscle showed 57% and 72% identity with rat UCP‐1 and UCP‐2. The mRNA was expressed abundantly in the skeletal muscle, moderately in the BAT, and slightly in the white adipose tissue (WAT) with a major band at 2.5 kb and a minor band at 2.8 kb, while the UCP‐2 gene expression was widely detected in the whole body with substantial levels in the WAT and with slight levels in the skeletal muscle and BAT. The rat UCP cloned in the present study showed 86% identity with the recently cloned human UCP‐3, which was also expressed abundantly in the skeletal muscle with a signal of 2.4 kb. Therefore, the rat UCP was considered to be rat UCP‐3. In rats fed high‐fat diet the UCP‐3 gene expression was augmented 2‐fold in the skeletal muscle while UCP‐2 mRNA levels were increased significantly (1.6‐fold) in the epididymal WAT. Augmented expression of UCPs may provide defense against high‐fat induced obesity and impairment of glucose metabolism.

Oxidized LDL Regulates Vascular Endothelial Growth Factor Expression in Human Macrophages and Endothelial Cells Through Activation of Peroxisome Proliferator–Activated Receptor-γ

Abstract— Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPAR&ggr; may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-(S)-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPAR&ggr; ligands. Therefore, we investigated the involvement of PPAR&ggr; in the regulation of VEGF by Ox-LDL. PPAR&ggr; expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 &mgr;g/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPAR&ggr; activators, troglitazone (TRO), and 15-deoxy-&Dgr;12,14-prostaglandin J2 (PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPAR&ggr; expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPAR&ggr; (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPAR&ggr;.

Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMP/cGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPs/cGMP/cGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk1/2 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPs/cGMP/cGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration.

Objective— We demonstrated previously that mouse embryonic stem (ES) cell–derived vascular endothelial growth factor receptor-2 (VEGF-R2)–positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration. Methods and Results— Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)–negative population emerged. These VEGF-R2-positive but tumor rejection antigen 1-60–negative cells were also positive for platelet-derived growth factor receptor &agr; and &bgr; chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and &agr;-smooth muscle actin+ mural cell. VE-cadherin+ cells, which were also CD34+ and VEGF-R2+ and thought to be endothelial cells in the early differentiation stage, could be expanded while maintaining their maturity. Their transplantation to the hindlimb ischemia model of immunodeficient mice contributed to the construction of new blood vessels and improved blood flow. Conclusions— We could identify the differentiation process from human ES cells to vascular cell components and demonstrate that expansion and transplantation of vascular cells at the appropriate differentiation stage may constitute a novel strategy for vascular regenerative medicine.

Down regulation of peroxisome proliferator-activated receptorγ expression by inflammatory cytokines and its reversal by thiazolidinediones

Aims/hypothesis. Previous studies show that inflammatory cytokines play a part in the development of insulin resistance. Thiazolidinediones were developed as insulin-sensitizing drugs and are ligands for the peroxisome proliferator-activated receptorγ (PPARγ). We hypothesized that the anti-diabetic mechanism of thiazolidinediones depends on the quantity of PPARγ in the insulin resistant state in which inflammatory cytokines play a part. Methods. We isolated rat PPARγ1 and γ2 cDNAs and examined effects of various cytokines and thiazolidinediones on PPARγ mRNA expression in rat mature adipocytes. Results. Various inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6 and leukaemia inhibitory factor decreased PPARγ mRNA expression. In addition, hydrogen peroxide, lysophosphatidylcholine or phorbol 12-myristate 13-acetate also decreased the expression of PPARγ. The suppression of PPARγ mRNA expression caused by 10 nmol/l of TNF-α was reversed 60 % and 55 % by treatment with 10–4 mol/l of troglitazone and 10–4 mol/l of pioglitazone, respectively. The suppression of glucose transporter 4 mRNA expression caused by TNF-α was also reversed by thiazolidinediones. Associated with the change of PPARγ mRNA expression, troglitazone improved glucose uptake suppressed by TNF-α. Conclusion/interpretation. Our study suggests that inflammatory cytokines could be factors that regulate PPARγ expression for possible modulation of insulin resistance. In addition, we speculate that the regulation of PPARγ mRNA expression may contribute to the anti-diabetic mechanism of thiazolidinediones. [Diabetologia (1999) 42: 702–710]

Thiazolidinediones, peroxisome proliferator-activated receptor γ agonists, regulate endothelial cell growth and secretion of vasoactive peptides

Insulin resistance has been highlighted as a common causal factor for glucose intolerance, hypertension and dyslipidemia, all of which are cardiovascular risk factors. A new class of antidiabetic agents, thiazolidinediones (TZDs), has been developed and demonstrated to improve insulin sensitivity. TZDs are high affinity ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the crucial transcription factor for adipocytes. Recent studies showed that PPARgamma is also expressed in monocytes/macrophages and is suggested to be involved in atherosclerosis. We could detect PPARgamma gene transcript in several cultured endothelial cells (human aortic endothelial cells (HAoECs), human coronary artery endothelial cells (HCAECs), human umbilical vein endothelial cells (HUVECs) and bovine carotid artery endothelial cells (BAECs)) as well as human coronary arteries we examined. Since endothelial dysfunction is critical for atherosclerosis, we investigated the effects of TZDs, troglitazone (TRO) and pioglitazone (PIO), on endothelial cell growth and secretion of C-type natriuretic peptide (CNP), which we demonstrated as a novel endothelium-derived relaxing peptide, and endothelin (ET), a potent vasoconstrictor, using HAoECs, HCAECs, HUVECs and BAECs. When all these cultured endothelial cells were daily treated with TRO and PIO for 5 days, both TRO and PIO (10(-8)M) significantly stimulated (3)H-thymidine incorporation of all these endothelial cells. In contrast, higher dose of TRO and PIO (10(-5)M) significantly suppressed DNA synthesis. TRO and PIO also exerted the compatible effect on the increase of cell numbers. TRO and PIO significantly enhanced CNP secretion from BAECs. In contrast, ET secretion from BAECs was suppressed by both TRO and PIO in a dose-dependent manner. The results of the present study suggest that TZDs modulate endothelial functions, including regulation of endothelial cell growth and secretion of endothelium-derived vasoactive substances, which affect vascular tone and remodeling in the process of atherosclerosis.

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration. Methods and Results After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity. Conclusions Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.

Accelerated Reendothelialization With Suppressed Thrombogenic Property and Neointimal Hyperplasia of Rabbit Jugular Vein Grafts by Adenovirus-Mediated Gene Transfer of C-Type Natriuretic Peptide

Background—Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft. Methods and Results—Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery. Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days. More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81±0.05 versus 0.30±0.14, P <0.01) and at 28 days (0.96±0.01 versus 0.45±0.08, P <0.001). The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts. Neointimal thickening was significantly suppressed in the Ad.CNP group. The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration. Conclusions—Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.