Abstract-Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-1 ϩ/Ϫ ) transgenic mice were subjected to Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured by DNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic hearts subjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.8Ϯ0.3% compared with 4.6Ϯ1.1% for wild-type controls (nϭ4; PϽ0.03). Phosphorylation of the protein kinase Akt/protein kinase B was elevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion, remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.6Ϯ0.3-fold). Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusionmediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activated during ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10 mol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-type and transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis in transgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and prevents apoptosis in hearts that overexpress the IGF-1 transgene. (Circ Res. 2001;88:609-614.) Key Words: ischemia Ⅲ hypoxia Ⅲ phosphoinositol-3Ј-kinase Ⅲ p38 mitogen-activated protein kinase Ⅲ SB203580 P rotective and antiapoptotic properties of insulin-like growth factor-1 (IGF-1) have been demonstrated in different models of myocardial ischemia and infarction 1-4 as well as in isolated cardiac myocytes subjected to ischemic or oxidative stress. 5,6 IGF-1 stimulates the phosphoinositol-3Ј (PI3)-kinase pathway, producing phosphoinositides that promote activation of the kinase Akt. 7,8 Activated Akt kinase plays a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins, 9 caspase 9, 10 and Fas ligand. 11 Transfer of mutationally activated PI3-kinase and Akt genes has been shown to prevent apoptosis of cardiac myocytes in vitro, 6 and activated Akt delivered by an adenovirus vector reduced apoptosis in the intact heart subjected to ischemia and reperfusion (I/R). 4 We previously reported that Igf-1 ϩ/Ϫ transgenic mice overexpressing IGF-1 had reduced rates of necrosis and apoptosis after myocardial infarction caused by coronary artery ligation. 3 The same hearts were also resistant to necrosis but not apoptosis caused by nonocclusive coronary artery constriction. 12 Other work has shown that the endogenous PI3-kinase pathway is activated in the postinfarcted myocardium, where it may contribute to cell survival and hypertrophy. [13][14][15] Regulation of the endogenous PI3-kinase pathway during I/R has not b...

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1␣, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1␣. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex.

EC-SOD overexpression to the lung ameliorated MCT-induced PH in rats. We suggest that EC-SOD may act as an antioxidant in PH and that increased oxidative stress may be important in the pathogenesis of MCT-induced PH.

We previously reported that whereas systemic continuous infusion of parathyroid hormone (PTH) accelerated orthodontic tooth movement, systemic but intermittent injection of PTH did not increase the rate of tooth movement. Analysis of these data suggested that continuous administration of PTH could be applicable for orthodontic therapy. In the present study, we investigated whether local and chronic application of PTH(1-34) would accelerate orthodontic tooth movement. To increase the residence time of PTH in the injected area, we used methylcellulose (MC) gel (2% W/V) for a slow-release formulation of PTH. MC gel containing PTH (PTH-MC) continuously released biologically active PTH into the acceptor medium for more than 72 hrs in vitro. When male rats received a local injection of PTH-MC into the subperiosteum in the mesio-palatal region of the maxillary first molar (M1) every other day, M1 movement, which was mesially drawn by an orthodontic coil spring attached to the maxillary incisors, was accelerated in a dose-dependent manner. PTH-MC injection at 1 microg/400 g body weight caused a 1.6-fold increase in the rate of tooth movement. The acceleration of tooth movement by PTH-MC injection was marked on days 6, 9, and 12. Local injection of PTH dissolved in saline without MC did not significantly accelerate tooth movement on day 6 or later. Histological examination revealed active osteoclastic bone resorption and a widened periodontal space on the compression side of the periodontal tissue in the PTH-MC-injected rats. These results suggest that local injection of PTH in a slow-release formulation is applicable to orthodontic therapy.