Background-Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, that may also cause a positive energy balance by stimulating food intake and inducing adiposity. We sought to investigate the pathophysiology of ghrelin in the cachexia associated with chronic heart failure (CHF). Methods and Results-Plasma ghrelin was measured in 74 patients with CHF and 12 control subjects, together with potentially important anabolic and catabolic factors, such as GH and tumor necrosis factor (TNF-␣). Patients with CHF were divided into two groups, those with cachexia (nϭ28) and those without cachexia (nϭ46). Plasma ghrelin did not significantly differ between all CHF patients and controls (181Ϯ10 versus 140Ϯ14 fmol/mL, PϭNS). However, plasma ghrelin was significantly higher in CHF patients with cachexia than in those without cachexia (237Ϯ18 versus 147Ϯ10 fmol/mL, PϽ0.001). Circulating GH, TNF-␣, norepinephrine, and angiotensin II were also significantly higher in CHF patients with cachexia than in those without cachexia. Interestingly, plasma ghrelin correlated positively with GH (rϭ0.28, PϽ0.05) and TNF-␣ (rϭ0.31, PϽ0.05) and negatively with body mass index (rϭϪ0.35, PϽ0.01). Conclusions-Plasma ghrelin was elevated in cachectic patients with CHF, associated with increases in GH and TNF-␣ and a decrease in body mass index. Considering ghrelin-induced positive energy effects, increased ghrelin may represent a compensatory mechanism under catabolic-anabolic imbalance in cachectic patients with CHF.

Background-Circulating endothelial progenitor cells (EPCs) migrate to injured vascular endothelium and differentiate into mature endothelial cells. We investigated whether transplantation of vasodilator gene-transduced EPCs ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Methods and Results-We obtained EPCs from cultured human umbilical cord blood mononuclear cells and constructed plasmid DNA of adrenomedullin (AM), a potent vasodilator peptide. We used cationic gelatin to produce ionically linked DNA-gelatin complexes. Interestingly, EPCs phagocytosed plasmid DNA-gelatin complexes, which allowed nonviral, highly efficient gene transfer into EPCs. Intravenously administered EPCs were incorporated into the pulmonary vasculature of immunodeficient nude rats given MCT. Transplantation of EPCs alone modestly attenuated MCT-induced pulmonary hypertension (16% decrease in pulmonary vascular resistance). Furthermore, transplantation of AM DNA-transduced EPCs markedly ameliorated pulmonary hypertension in MCT rats (39% decrease in pulmonary vascular resistance). MCT rats transplanted with AM-expressing EPCs had a significantly higher survival rate than those given culture medium or EPCs alone. Conclusions-Umbilical cord blood-derived EPCs had a phagocytosing action that allowed nonviral, highly efficient gene transfer into EPCs. Transplantation of AM gene-transduced EPCs caused significantly greater improvement in pulmonary hypertension in MCT rats than transplantation of EPCs alone. Thus, a novel hybrid cell-gene therapy based on the phagocytosing action of EPCs may be a new therapeutic strategy for the treatment of pulmonary hypertension. Key Words: pulmonary heart disease Ⅲ natriuretic peptides Ⅲ gene therapy Ⅲ endothelium T he pulmonary endothelium plays an important role in the regulation of pulmonary vascular tone through the release of vasoactive substances such as nitric oxide, prostacyclin, and adrenomedullin (AM). 1 Dysfunction of the endothelium may play a role in the pathogenesis of pulmonary hypertension, including primary pulmonary hypertension. 2 Thus, pulmonary endothelial cells may be a therapeutic target for the treatment of pulmonary hypertension. Recently, endothelial progenitor cells (EPCs) have been discovered in adult peripheral blood. 3 EPCs are mobilized from bone marrow into the peripheral blood in response to tissue ischemia or traumatic injury, migrate to sites of injured endothelium, and differentiate into mature endothelial cells in situ. 4 -6 These findings raise the possibility that transplanted EPCs may serve not only as a tissue-engineering tool to reconstruct the pulmonary vasculature but also as a vehicle for gene delivery to injured pulmonary endothelium.We prepared biodegradable gelatin that could hold negatively charged protein or plasmid DNA in its positively charged lattice structure. 7,8 We have shown that the gelatin is promptly phagocytosed and then gradually degraded by phagocytes, including macrophages. 9 However, whether EPCs phagocytose ionically l...

Ghrelin is an acylated peptide with growth-hormonereleasing activity (1 ). It was first isolated from rat and human stomach during the search for an endogenous ligand to the "orphan" G-protein-coupled receptor, growth hormone secretagogue receptor (2 ). The peptide contains 28 amino acids, and n-octanoylation of the Ser-3 hydroxyl group is necessary for biological activity. Most studies have focused on the somatotropic and orexigenic roles of ghrelin; therefore, little is known about the kinetics of this peptide. Because the ester bond is both chemically and enzymatically unstable, elimination of the octanoyl modification of ghrelin can occur during storage, handling, and/or dissolution in culture medium (3 ). Because of increased interest in ghrelin measurements, a standardized method of sample collection is required.In the present study, which focused on the active form of ghrelin, we investigated the effects of anticoagulants and storage conditions on ghrelin stability. To distinguish the active form of ghrelin, we established two ghrelinspecific RIAs; N-RIA recognizes the N-terminal, octanoylmodified portion of the peptide, whereas C-RIA recognizes the C-terminal portion. Thus, the value determined by N-RIA specifically measures active ghrelin, whereas the value determined by C-RIA gives the total ghrelin immunoreactivity, including both active and desacyl ghrelin (4 -6 ). The minimum detectable quantities in the N-and C-RIAs were 5.0 and 50 pmol/L, respectively. The respective intra-and interassay CV were 3% and 6% for the N-RIA and 6% and 9% for the C-RIA (n ϭ 8 assays). Data are reported as the mean (SD). Comparisons of the time course of ghrelin concentrations between subgroups were made by two-way ANOVA for repeated measures, followed by the Scheffé test. P Ͻ0.05 was considered statistically significant.All blood samples were taken from three healthy male volunteers who gave written informed consent. Blood was taken from the forearm vein and immediately divided into tubes for serum and plasma preparation using (a) disodium EDTA (1 g/L) with aprotinin (500 000 kIU/ L), (b) disodium EDTA alone, (c) heparin sodium, or (d) no anticoagulant. Synthetic human ghrelin was added to each blood sample at a final concentration of 40 g/L; each sample was then sequentially divided into two aliquots for incubation at either 4 or 37°C. After incubation for 0, 30, and 60 min, blood samples were centrifuged, diluted 1:200 in RIA buffer, and subjected to ghrelin-specific RIAs. A comparison of the effects of different anticoagulants on the detected ghrelin concentrations is shown in Table 1A. Although the serum and three different plasma samples tested gave comparable results for total ghrelin by C-RIA, the N-RIA gave ghrelin concentrations that were significantly decreased at 37°C. When the ghrelin was measured by N-RIA, serum samples were highly affected by such treatment; samples stored for 60 min at 37°C lost ϳ35% of the ghrelin compared with the basal values at 0 min (P Ͻ0.05). The ghrelin concentrations in samples co...

These data suggest that CNP may be useful as a novel antiremodeling agent.