Interaction modes of human orexin 2 receptor with selective and nonselective antagonists studied by NMR spectroscopy.

Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex.

Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.

Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R).

The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.

Cryo-EM structure of the human MT1-Gi signaling complex.

Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.

Vibrational analysis of acetylcholine binding to the M2 receptor.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm-1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.

Ligand Binding-Induced Structural Changes in the M2 Muscarinic Acetylcholine Receptor Revealed by Vibrational Spectroscopy.

M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine and mediates various cellular responses in the nervous system. Here, we used attenuated total reflection-Fourier transform infrared spectroscopy analyses on M2R reconstituted in a lipid membrane to understand the molecular mechanism behind the ligand binding-induced conformational changes. Upon agonist binding, M2R shows large spectral change of the amide-I band corresponding to backbone C═O stretch, which likely connects with the receptor activation in the lipid environment. These results pave the way to probe effects of different ligand binding on GPCRs using vibrational spectroscopy.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA.

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.

Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.

BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.

Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination.

G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.

Structure of the human histamine H1 receptor complex with doxepin.

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 191(5.39) and/or Lys 179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.

BACKGROUND: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely ß2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. RESULTS: The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. CONCLUSION: Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Cyclic GMP kinase and RhoA Ser188 phosphorylation integrate pro- and antifibrotic signals in blood vessels.

Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential. cGK I(+/-) mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoA(A188) in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent with the notion that RhoA(A188) escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoA(A188) became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated with vascular malfunction, predisposes individuals to vascular fibrosis.

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.

Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

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.

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.

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.

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.