Kazutoshi Miyashita

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.

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.

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.

Different Differentiation Kinetics of Vascular Progenitor Cells in Primate and Mouse Embryonic Stem Cells

Background—We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells. Methods and Results—In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2. By culturing these undifferentiated ES cells for 4 days on OP9 feeder layer, VEGF-R2 expression disappeared, and then reappeared after 8 days of differentiation. We then isolated these VEGF-R2–positive and vascular endothelial cadherin (VEcadherin)-negative cells by flow cytometry sorting. Additional 5-day reculture of these VEGF-R2+ VEcadherin− cells on OP9 feeder layer resulted in the appearance of platelet endothelial cell adhesion molecule-1 (PECAM1)-positive, VEcadherin-positive, endothelial nitric oxide synthase (eNOS)-positive endothelial cells. On a collagen IV-coated dish in the presence of serum, these cells differentiated into smooth muscle actin (SMA)-positive and calponin-positive mural cells (pericytes or vascular smooth muscle cells). Addition of 50ng/mL VEGF to the culture on a collagen IV-coated dish resulted in the appearance of PECAM1+ cells surrounded by SMA+ cells. In addition, these differentiated VEGF-R2+ cells can form tube-like structures in a 3-dimensional culture. Conclusion—Our findings indicate that differentiation kinetics of VPCs derived from primate and mouse ES cells were different. Differentiated VEGF-R2+ VEcadherin− cells can act as VPCs in primates. To seek the clinical potential of VPCs for vascular regeneration, investigations of primate ES cells are indispensable.

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3‐kinase (PI3K). AM promoted re‐endothelialization in vitro of wounded monolayer of HUVECs and neo‐vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA‐ and PI3K‐dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.

Angiotensin II Reduces Mitochondrial Content in Skeletal Muscle and Affects Glycemic Control

OBJECTIVE—Blockade of angiotensin (Ang) II has been shown to prevent new-onset type 2 diabetes. We focused on the effects of AngII on muscle mitochondria, especially on their biogenesis, as an underlining mechanism of type 2 diabetes. RESEARCH DESIGN AND METHODS—C2C12 cells and C57bl/6 mice were used to examine roles for AngII in the regulation of muscle mitochondria and to explore whether the effect was mediated by type 1 AngII receptor (AT1R) or type 2 receptor (AT2R). RESULTS—C2C12 cells treated with 10−8–10−6 mol/l AngII reduced the mitochondrial content associated with downregulation of the genes involved in mitochondrial biogenesis. The action of AngII was diminished by blockade of AT2R but not AT1R, whereas overexpression of AT2R augmented the effect. AngII increased mitochondrial ROS and decreased mitochondrial membrane potential, and these effects of AngII were significantly suppressed by blockade of either AT1R or AT2R. Chronic AngII infusion in mice also reduced muscle mitochondrial content in association with increased intramuscular triglyceride and deteriorated glycemic control. The AngII-induced reduction in muscle mitochondria in mice was partially, but significantly, reversed by blockade of either AT1R or AT2R, associated with increased fat oxidation, decreased muscle triglyceride, and improved glucose tolerance. Genes involved in mitochondrial biogenesis were decreased via AT2R but not AT1R under these in vivo conditions. CONCLUSIONS—Taken together, these findings imply the novel roles for AngII in the regulation of muscle mitochondria and lipid metabolism. AngII reduces mitochondrial content possibly through AT1R-dependent augmentation of their degradation and AT2R-dependent direct suppression of their biogenesis.

Angiogenic and Vasoprotective Effects of Adrenomedullin on Prevention of Cognitive Decline After Chronic Cerebral Hypoperfusion in Mice

Background and Purpose— Although subcortical vascular dementia, the major subtype of vascular dementia, is caused by a disruption in white matter integrity after cerebrovascular insufficiency, no therapy has been discovered that will restore cerebral perfusion or functional cerebral vessels. Because adrenomedullin (AM) has been shown to be angiogenic and vasoprotective, the purpose of the study was to investigate whether AM may be used as a putative treatment for subcortical vascular dementia. Methods— A model of subcortical vascular dementia was reproduced in mice by placing microcoils bilaterally on the common carotid arteries. Using mice overexpressing circulating AM, we assessed the effect of AM on cerebral perfusion, cerebral angioarchitecture, oxidative stress, white matter change, cognitive function, and brain levels of cAMP, vascular endothelial growth factor, and basic fibroblast growth factor. Results— After bilateral common carotid artery stenosis, mice overexpressing circulating AM showed significantly faster cerebral perfusion recovery due to substantial growth of the capillaries, the circle of Willis, and the leptomeningeal anastomoses and reduced oxidative damage in vascular endothelial cells compared with wild-type mice. Vascular changes were preceded by upregulation of cAMP, vascular endothelial growth factor, and basic fibroblast growth factor. White matter damage and working memory deficits induced by bilateral common carotid artery stenosis were subsequently restored in mice overexpressing circulating AM. Conclusions— These data indicate that AM promotes arteriogenesis and angiogenesis, inhibits oxidative stress, preserves white matter integrity, and prevents cognitive decline after chronic cerebral hypoperfusion. Thus, AM may serve as a strategy to tackle subcortical vascular dementia.

Transplantation of vascular cells derived from human embryonic stem cells contributes to vascular regeneration after stroke in mice.

BackgroundWe previously demonstrated that vascular endothelial growth factor receptor type 2 (VEGF-R2)-positive cells induced from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) and these vascular cells construct blood vessel structures in vitro. Recently, we have also established a method for the large-scale expansion of ECs and MCs derived from human ES cells. We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.MethodsPhosphate buffered saline, human peripheral blood mononuclear cells (hMNCs), ECs-, MCs-, or the mixture of ECs and MCs derived from human ES cells were intra-arterially transplanted into mice after transient middle cerebral artery occlusion (MCAo).ResultsTransplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes. The cerebral blood flow and the vascular density in the ischemic striatum on day 28 after MCAo had significantly improved in ECs-, MCs- and ECs+MCs-transplanted mice compared to that of mice injected with saline or transplanted with hMNCs. Moreover, compared to saline-injected or hMNC-transplanted mice, significant reduction of the infarct volume and of apoptosis as well as acceleration of neurological recovery were observed on day 28 after MCAo in the cell mixture-transplanted mice.ConclusionTransplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

Coenzyme Q10 reverses mitochondrial dysfunction in atorvastatin-treated mice and increases exercise endurance

Statins are cholesterol-lowering drugs widely used in the prevention of cardiovascular diseases; however, they are associated with various types of myopathies. Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and thus decrease biosynthesis of low-density lipoprotein cholesterol and may also reduce ubiquinones, essential coenzymes of a mitochondrial electron transport chain, which contain isoprenoid residues, synthesized through an HMG-CoA reductase-dependent pathway. Therefore, we hypothesized that statin treatment might influence physical performance through muscular mitochondrial dysfunction due to ubiquinone deficiency. The effect of two statins, atorvastatin and pravastatin, on ubiquinone content, mitochondrial function, and physical performance was examined by using statin-treated mice. Changes in energy metabolism in association with statin treatment were studied by using cultured myocytes. We found that atorvastatin-treated mice developed muscular mitochondrial dysfunction due to ubiquinone deficiency and a decrease in exercise endurance without affecting muscle mass and strength. Meanwhile, pravastatin at ten times higher dose of atorvastatin had no such effects. In cultured myocytes, atorvastatin-related decrease in mitochondrial activity led to a decrease in oxygen utilization and an increase in lactate production. Conversely, coenzyme Q(10) treatment in atorvastatin-treated mice reversed atorvastatin-related mitochondrial dysfunction and a decrease in oxygen utilization, and thus improved exercise endurance. Atorvastatin decreased exercise endurance in mice through mitochondrial dysfunction due to ubiquinone deficiency. Ubiquinone supplementation with coenzyme Q(10) could reverse atorvastatin-related mitochondrial dysfunction and decrease in exercise tolerance.