The role of mineralocorticoid receptor expression in brain remodeling after cerebral ischemia.

Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse 20-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.

Therapeutic potential of atrial natriuretic peptide administration on peripheral arterial diseases.

Peripheral arterial diseases are caused by arterial sclerosis and impaired collateral vessel formation, which are exacerbated by diabetes, often leading to leg amputation. We have reported that an activation of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway accelerated vascular regeneration and blood flow recovery in murine legs, for which ischemia had been induced by a femoral arterial ligation as a model for peripheral arterial diseases. In this study, ip injection of carperitide, a human recombinant atrial natriuretic peptide, accelerated blood flow recovery with increasing capillary density in ischemic legs not only in nondiabetic mice but also in mice kept upon streptozotocin-induced hyperglycemia for 16 wk, which significantly impaired the blood flow recovery compared with nondiabetic mice. Based on these findings, we tried to apply the administration of carperitide to the treatment of peripheral arterial diseases. The study group comprised a continuous series of 13 patients with peripheral arterial diseases (Fontaine's classification I, one; II, five; III, two; and IV, five), for whom conventional therapies had not accomplished appreciable results. Carperitide was administrated continuously and intravenously for 2 wk to Fontaine's class I-III patients and for 4 weeks to class IV patients. The dose was gradually increased to the maximum, with the patient's systolic blood pressure being kept above 100 mm Hg. Carperitide administration improved the ankle-brachial pressure index, intermittent claudication, rest pain, and ulcers. In conclusion, this study showed a therapeutic potential of carperitide to treat peripheral arterial diseases refractory to conventional therapies.

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

BACKGROUND: We 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. METHODS: Phosphate 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). RESULTS: Transplanted 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. CONCLUSION: Transplantation 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.

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 alpha and beta chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and alpha-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.

The neuroprotective and vasculo-neuro-regenerative roles of adrenomedullin in ischemic brain and its therapeutic potential.

Adrenomedullin (AM) is a vasodilating hormone secreted mainly from vascular wall, and its expression is markedly enhanced after stroke. We have revealed that AM promotes not only vasodilation but also vascular regeneration. In this study, we focused on the roles of AM in the ischemic brain and examined its therapeutic potential. We developed novel AM-transgenic (AM-Tg) mice that overproduce AM in the liver and performed middle cerebral artery occlusion for 20 min (20m-MCAO) to examine the effects of AM on degenerative or regenerative processes in ischemic brain. The infarct area and gliosis after 20m-MCAO was reduced in AM-Tg mice in association with suppression of leukocyte infiltration, oxidative stress, and apoptosis in the ischemic core. In addition, vascular regeneration and subsequent neurogenesis were enhanced in AM-Tg mice, preceded by increase in mobilization of CD34(+) mononuclear cells, which can differentiate into endothelial cells. The vasculo-neuro-regenerative actions observed in AM-Tg mice in combination with neuroprotection resulted in improved recovery of motor function. Brain edema was also significantly reduced in AM-Tg mice via suppression of vascular permeability. In vitro, AM exerted direct antiapoptotic and neurogenic actions on neuronal cells. Exogenous administration of AM in mice after 20m-MCAO also reduced the infarct area, and promoted vascular regeneration and functional recovery. In summary, this study suggests the neuroprotective and vasculo-neuro-regenerative roles of AM and provides basis for a new strategy to rescue ischemic brain through its multiple hormonal actions.

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK).

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.

Therapeutic potential of thiazolidinediones in activation of peroxisome proliferator-activated receptor gamma for monocyte recruitment and endothelial regeneration.

Thiazolidinediones, a new class of antidiabetic drugs that increase insulin sensitivity, have been shown to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Recent studies demonstrating that PPARgamma occurs in macrophages have focused attention on its role in macrophage functions. In this study, we investigated the effect of thiazolidinediones on monocyte proliferation and migration in vitro and the mechanisms involved. In addition, we examined the therapeutic potentials of thiazolidinediones for injured atherosclerotic lesions. Troglitazone and pioglitazone, the two thiazolidinediones, as well as 15-deoxy-delta12,14-prostaglandin J2 inhibited in a dose-dependent manner the serum-induced proliferation of THP-1 (human monocytic leukemia cells) and of U937 (human monoblastic leukemia cells), which permanently express PPARgamma. These ligands for PPARgamma also significantly inhibited migration of THP-1 induced by monocyte chemoattractant protein-1 (MCP-1). Troglitazone and 15-deoxy-delta12,14-prostaglandin J2 significantly suppressed the mRNA expression of the MCP family-specific receptor CCR2 (chemokine CCR2 receptor) in THP-1 at the transcriptional level. Furthermore, troglitazone significantly inhibited MCP-1 binding to THP-1. Oral administration of troglitazone to Watanabe heritable hyperlipidemic (WHHL) rabbits after balloon injury suppressed acute recruitment of monocytes/macrophages and accelerated re-endothelialization. These results suggest that thiazolidinediones have therapeutic potential for the treatment of diabetic vascular complications.

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.

Adrenomedullin promotes proliferation and migration of cultured endothelial cells.

Adrenomedullin (AM) is a vasoactive hormone which exerts its action through cyclic adenosine monophosphate(cAMP) /cAMP-dependent protein kinase (PKA) cascade and intracellular Ca2+ mobilization. Recently, evidence has accumulated that AM plays a critical role in the regulation of vascular tone, remodeling and morphogenesis. And although numerous reports have examined the action of AM on cultured vascular cells, the results have not been consistent and have depended on the experimental conditions used. Accordingly, the purpose of this study was to clarify the effect of AM on the proliferation and migration of cultured endothelial cells. Our results revealed that AM promoted the growth and migration of endothelial cells (ECs). AM significantly promoted the proliferation of human umbilical vein endothelial cells (HUVECs) (56.0 +/- 8.7% over the controls at 10(-9) mol/l) and this stimulative effect was inhibited by two AM antagonists, AM(22-52) and calcitonin gene-related peptide (CGRP) (8-37). The number of HUVECs migrated to the lower surface of the transwell apparatus was also increased dose-dependently in the AM group (30.4 +/- 4.2% over the controls at 10(-7) mol/l), and this increase was suppressed by the two AM antagonists and by two PKA antagonists, adenosine 3'5'-cyclic monophosphorothioate Rp-isomer and myristoylated protein kinase A inhibitor amide 14-22. The promoting action of AM on endothelial migration was also suppressed by LY294002, an inhibitor for phosphatidylinositol 3-kinase, but not by N(G)-nitro-L-arginine-methyl ester (L-NAME), an antagonist for nitric oxide synthase (NOS). These results indicate that AM promotes proliferation and migration of ECs via a cAMP/PKA dependent pathway and lend support to the idea that AM exerts beneficial effects on vascular regeneration and might be used as a novel therapeutic strategy for patients with vascular disease.

New diagnostic procedure for primary aldosteronism: adrenal venous sampling under adrenocorticotropic hormone and angiotensin II receptor blocker--application to a case of bilateral multiple adrenal microadenomas.

Formerly, the incidence of primary aldosteronism (PA) among patients with hypertension was believed to be less than 1%. However, recent studies have suggested a much higher incidence of 6.59%-14.4% among such patients. These findings suggest that many cases of PA caused by small aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA) have not been properly diagnosed. To make a more accurate diagnosis in such cases, we developed a new diagnostic procedure for localization of PA, namely, adrenal venous sampling under continuous infusion of adrenocorticotropic hormone (ACTH) and administration of angiotensin II receptor blocker (AVS with ACTH and ARB). Here, we confirm the efficacy of this procedure in the case of a 37-year-old male suspected of having PA. The anticipated diagnosis of PA was based on the presence of hypokalemia, low plasma renin activity (PRA), elevated plasma aldosterone concentration (PAC) and left adrenal mass. However, AVS with ACTH and ARB revealed the presence of bilateral multiple adrenal microadenomas. In the new AVS method, neither ACTH nor the renin-angiotensin system (RAS) exert any influence on the plasma aldosterone level, and a more accurate aldosterone secretary state and a more accurate assessment of the aldosterone secretion of both adrenal glands can be recognized than by conventional AVS. Use of this new method should enable identification of additional cases of APA among patients diagnosed with essential hypertension.

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress.

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.

Significance of adrenocorticotropin stimulation test in the diagnosis of an aldosterone-producing adenoma.

CONTEXT: Adrenal venous sampling is the "gold standard" test in the diagnosis of an aldosterone-producing adenoma (APA) among patients with primary aldosteronism (PA) but is available only in specialized medical centers. Meanwhile, an APA is reported to be generally more sensitive to ACTH than idiopathic hyperaldosteronism. OBJECTIVE: The aim was to evaluate the diagnostic accuracy of the ACTH stimulation test in the diagnosis of an APA among those with suspicion of PA. PATIENTS AND SETTING: Fifty-nine patients admitted to Kyoto University Hospital on suspicion of PA were included in the study. INTERVENTIONS: ACTH stimulation tests with 1-mg dexamethasone suppression were performed. MAIN OUTCOME MEASURE: Plasma aldosterone concentrations (PAC) were examined every 30 min after ACTH stimulation. Receiver-operated characteristics curve analysis was used to evaluate the diagnostic accuracy. RESULTS: PAC after ACTH stimulations were significantly higher in patients with an APA than in patients with idiopathic hyperaldosteronism or non-PA. Receiver-operated characteristics curve analyses showed that the PAC after ACTH stimulation was effective for the diagnosis of an APA among patients suspected of PA. The diagnostic accuracy was highest at 90 min after ACTH injection, with the optimal cutoff value greater than 37.9 ng/dl corresponding with sensitivity and specificity of 91.3 and 80.6% for the diagnosis of an APA. CONCLUSIONS: Our study indicates that the ACTH stimulation test is useful in the diagnosis of an APA among patients suspected of PA. This test can be used to select patients who are highly suspected of an APA and definitely require adrenal venous sampling.

C-type natriuretic peptide as a new regulator of food intake and energy expenditure.

The physiological implication of C-type natriuretic peptide (CNP) including energy metabolism has not been elucidated, because of markedly short stature in CNP-null mice. In the present study we analyzed food intake and energy expenditure of CNP-null mice with chondrocyte-targeted CNP expression (CNP-Tg/Nppc(-/-) mice), in which marked skeletal dysplasia was rescued, to investigate the significance of CNP under minimal influences of skeletal phenotypes. In CNP-Tg/Nppc(-/-) mice, body weight and body fat ratio were reduced by 24% and 32%, respectively, at 20 wk of age, and decreases of blood glucose levels during insulin tolerance tests were 2-fold exaggerated at 17 wk of age, as compared with CNP-Tg/Nppc(+/+) mice. Urinary noradrenalin excretion of CNP-Tg/Nppc(-/-) mice was greater than that of CNP-Tg/Nppc(+/+) mice by 28%. In CNP-Tg/Nppc(-/-) mice, rectal temperature at 1600 h was higher by 1.1 C, and uncoupling protein-1 mRNA expression in the brown adipose tissue was 2-fold increased, which was canceled by propranolol administration, as compared with CNP-Tg/Nppc(+/+) mice. Oxygen consumption was significantly increased in CNP-Tg/Nppc(-/-) mice compared with that in CNP-Tg/Nppc(+/+) mice. Food intake of CNP-Tg/Nppc(-/-) mice upon ad libitum feeding and refeeding after 48 h starvation were reduced by 21% and 61%, respectively, as compared with CNP-Tg/Nppc(+/+) mice. This study unveiled a new aspect of CNP as a molecule regulating food intake and energy expenditure. Further analyses on precise mechanisms of CNP actions would lead to the better understanding of the significance of the CNP/guanylyl cyclase-B system in food intake and energy expenditure.

Sirt1 plays an important role in mediating greater functionality of human ES/iPS-derived vascular endothelial cells.

OBJECTIVE: We previously succeeded in inducing and isolating vascular endothelial cells (ECs) from both human embryonic stem (ES) and induced pluripotent stem (iPS) cells. Here, we compared the functionality of human adult ECs (HAECs), human ES-derived ECs (ESECs) and human iPS-derived ECs (iPSECs). METHODS AND RESULTS: We compared the cell proliferative potential, potential for migration, and tolerance to oxidative stress. ESECs were significantly superior to HAECs in all of these cell functions. The cell functions of iPSECs were comparable to those of ESECSs and also superior to HAECs. We then analyzed the gene expressions of HAECs, ESECs and iPSECs, and observed that the expression level of Sirt1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, is higher in ESECs and iPSECs than in HAECs. The inhibition of Sirt1 with a Sirt1-specific inhibitor and siRNA antagonized these differences between the three types of cells. CONCLUSIONS: Sirt1 plays a key role in the high cellular function of ESECs and iPSECs. Although further in vivo investigations are required, this study initially demonstrated the potential of ESECs and iPSECs as the cell source for regenerative medicine, and also showed the potential of ES cells as a useful tool for elucidating the molecular mechanism of cell aging.

Inhibition of hepatic damage and liver fibrosis by brain natriuretic peptide.

Anti-fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl(4))-induced liver fibrosis model using BNP-transgenic (Tg) and wild-type (WT) mice. Twice-a-week intraperitoneal injections of CCl(4) for 8 weeks resulted in massive liver fibrosis, augmented transforming growth factor (TGF)-beta(1) and type I procollagen alpha(1) chain (Col1a1) mRNA expression, and the hepatic stellate cell (HSC) activation in WT mice, all of which were significantly suppressed in Tg mice. These observations indicate that BNP inhibits liver fibrosis by attenuating the activation of HSCs.

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)-gamma coactivator (PGC)-1alpha and PPARdelta. 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-1alpha and PPARdelta 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.

Augmentation of neovascularization [corrected] 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.

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 cGMPcGMP-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 NPscGMPcGK 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 Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK 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.