Therapeutic angiogenesis for patients with no-option critical limb ischemia by adipose-derived regenerative cells: TACT-ADRC multicenter trial.

BACKGROUND: Patients with critical limb ischemia (CLI) still have a high rate of lower limb amputation, which is associated with not only a decrease in quality of life but also poor life prognosis. Implantation of adipose-derived regenerative cells (ADRCs) has an angiogenic potential for patients with limb ischemia. OBJECTIVES: We investigated safety, feasibility, and efficacy of therapeutic angiogenesis by cell transplantation (TACT) of ADRCs for those patients in multicenter clinical trial in Japan. METHODS: The TACT-ADRC multicenter trial is a prospective, interventional, open-labeled study. Patients with CLI (Fontaine class III-IV) who have no other option for standard revascularization therapy were enrolled in this study. Thirty-four target ischemic limbs of 29 patients were received freshly isolated autologous ADRCs implantation. RESULTS: The overall survival rate at a post-operative period and at 6 months follow-up was 100% at any time points. As a primary endpoint for efficacy evaluation, 32 limbs out of 34 (94.1%) were free from major amputation for 6 months. Numerical rating scale (from 6 to 1) as QOL score, ulcer size (from 317 mm2 at to 109 mm2), and 6-min walking distance (from 255 to 369 m) improved in 90.6%, 83.3%, and 72.2% patients, respectively. CONCLUSIONS: Implantation of autologous ADRCs could be safe and effective for the achievement of therapeutic angiogenesis in the multicenter settings, as a result in no major adverse event, optimal survival rate, and limb salvage for patients with no-conventional option against critical limb ischemia. TRN: jRCTb040190118; Date: Nov. 24th, 2015.

Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors.

It is widely accepted that adipose-derived regenerative cells (ADRCs) can differentiate into mesodermal lineage cells. However, reprogramming adult ADRCs into mature cardiomyocytes is challenging. We investigated the induction of myocardial differentiation in ADRCs via direct reprogramming using lentiviral gene transfer. First, we identified candidate transcriptional factors by performing RNA sequencing and ultimately confirmed that the combination of six unique factors (Baf60c, Gata4, Gata6, Klf15, Mef2a, and Myocd) could efficiently express enhanced green fluorescent protein (GFP) in ADRCs isolated from adult alpha-myosin heavy chain promoter-driven GFP transgenic mice. The GFP-positive ADRCs induced by six factors (6F-ADRCs) expressed multiple cardiac genes and revealed cardiac differentiation in bioinformatic analysis. Moreover, injection of 6F-ADRCs into acute myocardial infarcted tissues in vivo resulted in the improvement of survival rate, fractional shortening, and reduction of infarction scar area. This study provides an alternative method for direct reprogramming of adult ADRCs into cardiomyocytes.

Important Role of Concomitant Lymphangiogenesis for Reparative Angiogenesis in Hindlimb Ischemia.

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Therapeutic angiogenesis using autologous adipose-derived regenerative cells in patients with critical limb ischaemia in Japan: a clinical pilot study.

Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study-Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study-was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34+ and CD133+ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A165b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A165b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206+ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

Rationale and Design of Therapeutic Angiogenesis by Cell Transplantation Using Adipose-Derived Regenerative Cells in Patients With Critical Limb Ischemia　- TACT-ADRC Multicenter Trial.

Background: Despite the growing knowledge regarding optimal treatments for critical limb ischemia (CLI), there are still a considerable number of patients who have to undergo major limb amputation. Intramuscular injection of autologous adipose-derived regenerative cells (ADRCs) in these patients has shown therapeutic potential in improving tissue ischemia, in both preclinical and initial pilot studies. Here, we present a clinical protocol for ADRCs use in a multicenter trial. Methods and Results: The TACT-ADRC multicenter trial is a prospective, interventional, single-arm, open-labeled study at 8 hospitals in Japan, investigating the safety and feasibility of intramuscular injections of ADRCs and testing the hypothesis that this treatment promotes neovascularization and improves major amputation-free survival rates in patients with CLI who have no other treatment option. 40 patients with CLI will be enrolled and followed up from November 2015 to November 2020. Freshly isolated autologous ADRCs will be injected into the target ischemic limbs. Survival rate, adverse events, major limb amputation, ulcer size, 6-min walking distance, numerical rating scale, ankle-brachial pressure index, skin perfusion pressure and digital subtraction angiography will be evaluated at baseline and during 6 months' follow-up. Conclusions: This trial will demonstrate whether implantation of autologous ADRCs is a safe and effective method for therapeutic angiogenesis, resulting in an improvement in major amputation-free survival rates in patients with CLI.

Cardiomyocytes capture stem cell-derived, anti-apoptotic microRNA-214 via clathrin-mediated endocytosis in acute myocardial infarction.

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication that have the potential to improve cardiac function when used in cell-based therapy. However, the means by which cardiomyocytes respond to EVs remains unclear. Here, we sought to clarify the role of exosomes in improving cardiac function by investigating the effect of cardiomyocyte endocytosis of exosomes from mesenchymal stem cells on acute myocardial infarction (MI). Exposing cardiomyocytes to the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under hypoxia in vitro. In vivo, the injection of ADRCs into the heart simultaneous with coronary artery ligation decreased overall cardiac infarct area and prevented cardiac rupture after acute MI. Quantitative RT-PCR-based analysis of the expression of 35 known anti-apoptotic and secreted microRNAs (miRNAs) in ADRCs revealed that ADRCs express several of these miRNAs, among which miR-214 was the most abundant. Of note, miR-214 silencing in ADRCs significantly impaired the anti-apoptotic effects of the ADRC treatment on cardiomyocytes in vitro and in vivo To examine cardiomyocyte endocytosis of exosomes, we cultured the cardiomyocytes with ADRC-derived exosomes labeled with the fluorescent dye PKH67 and found that hypoxic culture conditions increased the levels of the labeled exosomes in cardiomyocytes. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, significantly suppressed the ADRC-induced decrease of hypoxia-damaged cardiomyocytes and also decreased hypoxia-induced cardiomyocyte capture of both labeled EVs and extracellular miR-214 secreted from ADRCs. Our results indicate that clathrin-mediated endocytosis in cardiomyocytes plays a critical role in their uptake of circulating, exosome-associated miRNAs that inhibit apoptosis.

Alteration in ventricular pressure stimulates cardiac repair and remodeling.

The mammalian heart undergoes complex structural and functional remodeling to compensate for stresses such as pressure overload. While studies suggest that, at best, the adult mammalian heart is capable of very limited regeneration arising from the proliferation of existing cardiomyocytes, how myocardial stress affects endogenous cardiac regeneration or repair is unknown. To define the relationship between left ventricular afterload and cardiac repair, we induced left ventricle pressure overload in adult mice by constriction of the ascending aorta (AAC). One week following AAC, we normalized ventricular afterload in a subset of animals through removal of the aortic constriction (de-AAC). Subsequent monitoring of cardiomyocyte cell cycle activity via thymidine analog labeling revealed that an acute increase in ventricular afterload induced cardiomyocyte proliferation. Intriguingly, a release in ventricular overload (de-AAC) further increases cardiomyocyte proliferation. Following both AAC and de-AAC, thymidine analog-positive cardiomyocytes exhibited characteristics of newly generated cardiomyocytes, including single diploid nuclei and reduced cell size as compared to age-matched, sham-operated adult mouse myocytes. Notably, those smaller cardiomyocytes frequently resided alongside one another, consistent with local stimulation of cellular proliferation. Collectively, our data demonstrate that adult cardiomyocyte proliferation can be locally stimulated by an acute increase or decrease of ventricular pressure, and this mode of stimulation can be harnessed to promote cardiac repair.

Impact of Therapeutic Angiogenesis Using Autologous Bone Marrow-Derived Mononuclear Cells Implantation in Critical Limb Ischemia With Scleroderma　- Subanalysis of the Long-Term Clinical Outcomes Survey.

BACKGROUND: Many patients with collagen disease (CD), particularly scleroderma (SSc), develop critical limb ischemia (CLI), which leads to limb amputation. However, conventional therapies, including revascularization via surgical bypass, showed poor outcomes in CLI patients with CD. Many CLI patients with SSc showed poor responses to combination therapies including intravenous iloprost, PDE-5 inhibitors, and bosentan. Therefore, new methods of improving the peripheral circulation for limb salvage are required. This study was a subanalysis of the long-term clinical outcomes after autologous bone marrow-derived mononuclear cells (BM-MNC) in CLI patients with SSc. Methods and Results: We assessed no-option CLI patients with CD who underwent BM-MNC implantation at 10 institutes; 69 patients (39 with SSc-related diseases (SSc group) and 30 with other CDs (non-SSc group)), were included. The median follow-up duration was 36.5 months. The 10-year overall survival rate was 59.1% in the SSc group and 82.4% in the non-SSc group. The 10-year major amputation-free rates were 97.4% and 82.6%, respectively. The number of major or minor amputations in the SSc group trended to be less than that in the non-SSc group. Significant improvements in visual analog scale scores were observed in both groups. CONCLUSIONS: The BM-MNC implantation may be feasible in no-option CLI patients with CD. In the SSc group, limb salvage rate tended to be higher than in the non-SSc group.

Long-Term Clinical Outcomes Survey of Bone Marrow-Derived Cell Therapy in Critical Limb Ischemia in Japan.

BACKGROUND: The Therapeutic Angiogenesis by Cell Transplantation (TACT) trial demonstrated the efficacy and safety of autologous bone marrow-derived mononuclear cells (BM-MNCs) in patients with critical limb ischemia (CLI). The present study aimed to assess the long-term clinical outcomes of therapeutic angiogenesis using autologous BM-MNC implantation under advanced medical treatment in Japan.Methods and Results:The study was retrospective, observational, and non-controlled. We assessed no-option CLI patients who had BM-MNC implantation performed in 10 institutes. Overall survival (OS), major amputation-free (MAF), and amputation-free survival (AFS) rates were primary endpoints of this study. The median follow-up duration was 31.7 months. The 10-year OS rate was 46.6% in patients with arteriosclerosis obliterans (ASO) (n=168), 90.5% in patients with thromboangiitis obliterans (TAO) (n=108), and 67.6% in patients with collagen disease-associated vasculitis (CDV) (n=69). The 10-year MAF rate was 70.1%, 87.9%, and 90.9%, respectively. The 10-year AFS rate was 37.8%, 80.9%, and 61.2%, respectively. Major adverse cardiovascular events occurred in 6.0% of patients with ASO, 1.9% of patients with TAO, and no patients with CDV. CONCLUSIONS: Therapeutic angiogenesis using autologous BM-MNC implantation may be feasible and safe in patients with no-option CLI, particularly those with CLI caused by TAO or CDV.

Balance between angiogenic and anti-angiogenic isoforms of VEGF-A is associated with the complexity and severity of coronary artery disease.

INTRODUCTION: Assessing the complexity of coronary artery disease (CAD) is clinically important. Vascular endothelial growth factor A (VEGF-A) is a powerful and the most important regulator of angiogenesis. It has been reported that the anti-angiogenic isoform of VEGF-A (VEGF-A165b) inhibits angiogenesis. The purpose of this study was to evaluate the relationship between the complexities of CAD using the Syntax score (SS) and the levels of circulating total VEGF-A or VEGF-A165b. METHODS: A total of 268 patients who underwent percutaneous coronary intervention (PCI) were enrolled. Of these, 73 patients without acute coronary syndrome or previous revascularization were included in this study. These subjects were divided into two groups according to the SS. Circulating total VEGF-A and VEGF-A165b levels were measured using an enzyme-linked immunosorbent assay. RESULTS: Circulating levels of total VEGF-A were significantly higher in the SS>22 (high SS) group than in the SS≤22 (low SS) group (p=0.018). Moreover, the ratio of VEGF-A165b to total VEGF-A was significantly lower for the high SS group (p=0.004). The levels of total VEGF-A independently predicted high SS after adjusting for other significant variables among patients who underwent PCI (odds ratio, 1.004; 95% CI, 1.001 to 1.006; p=0.01). CONCLUSIONS: These data indicated that high SS was associated with circulating levels of total VEGF-A and the ratio of VEGF-A165b to total VEGF-A in patients with complex CAD.

Diallyl Trisulfide Augments Ischemia-Induced Angiogenesis via an Endothelial Nitric Oxide Synthase-Dependent Mechanism.

BACKGROUND: Hydrogen sulfide (H2S) exerts beneficial actions against the development of cardiovascular disease. Diallyl trisulfide (DATS) is an organic polysulfide found in garlic oil that liberates H2S under physiological conditions. This study investigated whether DATS modulates endothelial cell function, as well as revascularization processes in a mouse model of hind-limb ischemia.Methods and Results:Wild-type (WT), endothelial nitric oxide synthase-deficient (eNOS-KO) and Akt1-heterogenic deficient (Akt-Het) mice were subjected to unilateral hindlimb ischemia (HLI). DATS or a vehicle control was injected into the abdomen of mice for up to 10 days following HLI induction. Treatment with DATS enhanced blood flow recovery and capillary density in the ischemic limbs of WT mice. This was accompanied by a reduction in apoptotic activity and oxidative stress in the ischemic muscles. DATS also increased the phosphorylation of Akt and eNOS in ischemic muscles. In contrast to WT mice, DATS did not improve blood flow of eNOS-KO and Akt-Het mice. In cultured human umbilical vein endothelium cells, DATS decreased apoptotic activity and oxidative stress under hypoxic conditions, and stimulated the phosphorylation of Akt and eNOS. Inhibition of Akt or NOS signaling reversed DATS-stimulated eNOS phosphorylation and blocked the effects of DATS on apoptosis and oxidative stress. CONCLUSIONS: These observations suggest that DATS promotes revascularization in response to HLI through its ability to stimulate the Akt-eNOS signaling pathway.

Soybean ß-Conglycinin Prevents Age-Related Hearing Impairment.

Obesity-related complications are associated with the development of age-related hearing impairment. ß-Conglycinin (ß-CG), one of the main storage proteins in soy, offers multiple health benefits, including anti-obesity and anti-atherosclerotic effects. Here, to elucidate the potential therapeutic application of ß-CG, we investigated the effect of ß-CG on age-related hearing impairment. Male wild-type mice (age 6 months) were randomly divided into ß-CG-fed and control groups. Six months later, the body weight was significantly lower in ß-CG-fed mice than in the controls. Consumption of ß-CG rescued the hearing impairment observed in control mice. Cochlear blood flow also increased in ß-CG-fed mice, as did the expression of eNOS in the stria vascularis (SV), which protects vasculature. ß-CG consumption also ameliorated oxidative status as assessed by 4-HNE staining. In the SV, lipofuscin granules of marginal cells and vacuolar degeneration of microvascular pericytes were decreased in ß-CG-fed mice, as shown by transmission electron microscopy. ß-CG consumption prevented loss of spiral ganglion cells and reduced the frequencies of lipofuscin granules, nuclear invaginations, and myelin vacuolation. Our observations indicate that ß-CG ameliorates age-related hearing impairment by preserving cochlear blood flow and suppressing oxidative stress.

The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.

RATIONALE: Despite 4 decades of intense effort and substantial financial investment, the cardioprotection field has failed to deliver a single drug that effectively reduces myocardial infarct size in patients. A major reason is insufficient rigor and reproducibility in preclinical studies. OBJECTIVE: To develop a multicenter, randomized, controlled, clinical trial-like infrastructure to conduct rigorous and reproducible preclinical evaluation of cardioprotective therapies. METHODS AND RESULTS: With support from the National Heart, Lung, and Blood Institute, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR), based on the principles of randomization, investigator blinding, a priori sample size determination and exclusion criteria, appropriate statistical analyses, and assessment of reproducibility. To validate CAESAR, we tested the ability of ischemic preconditioning to reduce infarct size in 3 species (at 2 sites/species): mice (n=22-25 per group), rabbits (n=11-12 per group), and pigs (n=13 per group). During this validation phase, (1) we established protocols that gave similar results between centers and confirmed that ischemic preconditioning significantly reduced infarct size in all species and (2) we successfully established a multicenter structure to support CAESAR's operations, including 2 surgical centers for each species, a Pathology Core (to assess infarct size), a Biomarker Core (to measure plasma cardiac troponin levels), and a Data Coordinating Center-all with the oversight of an external Protocol Review and Monitoring Committee. CONCLUSIONS: CAESAR is operational, generates reproducible results, can detect cardioprotection, and provides a mechanism for assessing potential infarct-sparing therapies with a level of rigor analogous to multicenter, randomized, controlled clinical trials. This is a revolutionary new approach to cardioprotection. Importantly, we provide state-of-the-art, detailed protocols ("CAESAR protocols") for measuring infarct size in mice, rabbits, and pigs in a manner that is rigorous, accurate, and reproducible.

Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism.

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production.

Multilayered adipose-derived regenerative cell sheets created by a novel magnetite tissue engineering method for myocardial infarction.

BACKGROUND: Adipose-derived regenerative cells (ADRCs) are a promising source of autologous stem cells for regeneration and repair of damaged tissue. Herein, we investigated the therapeutic potential of ADRC sheets created by a magnetite tissue engineering technology (Mag-TE) for myocardial infarction. METHODS AND RESULTS: Adipose tissue was obtained from wild-type (WT) mice and ADRCs were isolated. ADRCs incubated with magnetic nanoparticle-containing liposomes (MCLs) were cultured. MCL-labeled ADRCs were mixed with a diluted extracellular matrix (ECM) precursor, and a magnet was placed on the reverse side. Magnetized ADRCs formed multilayered cell sheets after a 24-h incubation. WT mice were subjected to myocardial infarction by permanent ligation of the left anterior descending artery. We then transplanted the constructed ADRC sheet or a cell-free collagen gel sheet, as a control, onto the infarcted myocardium using an Alnico magnet before skin closure. Cardiac parameters were measured by echocardiogram, and angiogenesis was determined by tissue capillary density. ADRC sheet-treated mice showed significant improvements in systolic function, infarct wall thinning, and fibrotic length after myocardial infarction. ADRC sheet implantation also promoted angiogenesis in both the infarct area and the border zone in WT mice after myocardial infarction. The angiogenic effects of ADRC sheets were attributed to an increased expression of VEGF and bFGF mRNA in ischemic hearts. CONCLUSIONS: ADRC sheets created by this Mag-TE method protect the heart against pathological cardiac remodeling. Our ADRC sheets have the potential to be a novel regenerative strategy for ischemic heart disease.

Therapeutic angiogenesis by autologous adipose-derived regenerative cells: comparison with bone marrow mononuclear cells.

Transplantation of adipose-derived regenerative cell (ADRC) enhances ischemia-induced angiogenesis, but the underlying mechanism remains unknown. Here, we compared the efficacy between ADRC and bone marrow mononuclear cell (BM-MNC) transplantation in rabbits model of hindlimb ischemia and examined the possible roles of alternative phenotypic macrophages polarization in ADRC-mediated angiogenesis using mice model of hindlimb ischemia. ADRCs and BM-MNCs were isolated from New Zealand White rabbits and C57BL/6J mice. In rabbit studies, our data showed that ADRCs could incorporate into the endothelial vasculature in vitro and in vivo. Both ADRC-conditioned media (CM) and BM-MNC-CM enhanced the migratory ability and interrupted the process of apoptosis in human umbilical vein endothelial cells. Four weeks after cell transplantation, augmentation of postnatal neovascularization was observed in the ischemic muscle injected with either ADRCs or BM-MNCs. In mice studies, we presented that ADRCs polarized into the IL-10-releasing M2 macrophages through PGE2-EP2/4 axis and suppressed the expressions of TNF-α and IL-6 in the ischemic muscle. Gene expressions of several angiogenic cytokines were amplified in the macrophages cultured in ADRC-CM rather than BM-MNC-CM. Blockade of IL-10 using neutralizing MAb attenuated the ADRC-mediated angiogenesis and caused muscle apoptosis in vivo. In conclusion, ADRC transplantation harvested similar effect of neovascularization augmentation compared with BM-MNC in experimental rabbit model of hindlimb ischemia; ADRC displayed a unique immunoregulatory manner of accelerating IL-10-releasing M2 macrophages polarization through the PGE2-EP2/4 axis.

Nitrite therapy improves left ventricular function during heart failure via restoration of nitric oxide-mediated cytoprotective signaling.

RATIONALE: Nitric oxide (NO) bioavailability is reduced in the setting of heart failure. Nitrite (NO2) is a critically important NO intermediate that is metabolized to NO during pathological states. We have previously demonstrated that sodium nitrite ameliorates acute myocardial ischemia/reperfusion injury. OBJECTIVE: No evidence exists as to whether increasing NO bioavailability via nitrite therapy attenuates heart failure severity after pressure-overload-induced hypertrophy. METHODS AND RESULTS: Serum from patients with heart failure exhibited significantly decreased nitrosothiol and cGMP levels. Transverse aortic constriction was performed in mice at 10 to 12 weeks. Sodium nitrite (50 mg/L) or saline vehicle was administered daily in the drinking water postoperative from day 1 for 9 weeks. Echocardiography was performed at baseline and at 1, 3, 6, and 9 weeks after transverse aortic constriction to assess left ventricular dimensions and ejection fraction. We observed increased cardiac nitrite, nitrosothiol, and cGMP levels in mice treated with nitrite. Sodium nitrite preserved left ventricular ejection fraction and improved left ventricular dimensions at 9 weeks (P<0.001 versus vehicle). In addition, circulating and cardiac brain natriuretic peptide levels were attenuated in mice receiving nitrite (P<0.05 versus vehicle). Western blot analyses revealed upregulation of Akt-endothelial nitric oxide-nitric oxide-cGMP-GS3Kß signaling early in the progression of hypertrophy and heart failure. CONCLUSIONS: These results support the emerging concept that nitrite therapy may be a viable clinical option for increasing NO levels and may have a practical clinical use in the treatment of heart failure.