Evolution of GADD34 expression after focal cerebral ischaemia.

GADD34, a stress response protein associated with cell rescue, DNA repair and apoptosis, is expressed in the ischaemic brain. The C-terminal region of GADD34 has homology with the Herpes Simplex Virus protein, ICP34.5, which overcomes the protein synthesis block after viral infection by actively dephosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). The carboxy terminus of GADD34 is also capable of dephosphorylating eIF2alpha and therefore has the capacity to restore the protein synthesis shutoff associated with ischaemia. This study examines the distribution and time course of GADD34 expression after focal cerebral ischaemia. Focal ischaemia or sham procedure was carried out on Sprague-Dawley rats with survival times of 4, 12, 24 h, 7 and 30 days. Brains were processed for histology and immunohistochemistry. Ischaemic damage was mapped onto line diagrams and GADD34 positive cells counted in selected regions of cortex and caudate. GADD34 immunopositive cells (mainly neurones), expressed as cells/mm2, were present in ischaemic brains at 4 h (e.g., peri-infarct cortex 20 +/- 5; contralateral cortex 3 +/- 1, P < 0.05). Of the time points examined, numbers of GADD34 positive cells were highest 24 h after ischaemia (peri-infarct cortex 31 +/- 7.3, contralateral cortex 0.1 +/- 0.1, P < 0.05). Immunopositive cells, following a similar time course, were identified within the peri-infarct zone in the caudate nucleus and in ipsilateral cingulate cortex (possibly as a consequence of cortical spreading depression). GADD34 positive cells did not co-localise with a marker of irreversible cell death (TUNEL). Taken together, GADD34 positive cells in key neuroanatomical locations pertinent to the evolving ischaemic lesion, the lack of co-localisation with TUNEL and the protein's known effects on restoring protein synthesis, repairing DNA and involvement in ischaemic pre-conditioning suggests that it has the potential to influence cell survival in ischaemically compromised tissue.

Myocardial expression of heat shock protein 70i protects early postoperative right ventricular function in cyanotic tetralogy of Fallot.

BACKGROUND: Right ventricular dysfunction occurs after tetralogy of Fallot repair and may relate to greater myocardial vulnerability to ischemia-reperfusion injury in cyanotic patients. The inducible form of heat shock protein 70 (HSP-70i), a molecular chaperone, is upregulated in response to cellular stress and limits myocardial injury against ischemia-reperfusion. We evaluated the myocardial expression of HSP-70i and its relation to right ventricular function and clinical outcome in patients with tetralogy of Fallot undergoing corrective surgery. METHODS: Twenty patients with tetralogy of Fallot were studied: 10 cyanotic (group Cy) and 10 noncyanotic (group noCy). Western blot was used to quantify HSP-70i from resected right ventricular outflow tract myocardium at baseline and subsequent ischemic time. Biventricular function was quantified by tissue Doppler echocardiography and compared with that of 15 age-matched healthy children. Postoperative systemic perfusion was assessed by mixed venous oxygen saturation, oxygen extraction ratio, and lactate. RESULTS: Group Cy had thicker septum (median 0.85 vs 0.66 cm; P = .01) and longer crossclamp time (median 100.0 vs 67.5 minutes; P = .004). There were no difference in HSP-70i between groups at baseline (4.12 vs 3.44 relative optical density; P = .45) or subsequent ischemic time. Preoperative biventricular systolic function was reduced in patients with tetralogy compared with controls with further postoperative right ventricular impairment. Group Cy had higher troponin-I levels (median 16.5 vs 11.1 ng/mL; P = .04) and inotrope scores (14.0 vs 6.5; P = .05) but no differences in ventricular function, mixed venous oxygen saturation, oxygen extraction ratio, and lactate between groups. In group Cy, baseline HSP-70i correlated with better postoperative right ventricular function (rho = 0.80; P = .009), mixed venous oxygen saturation (rho = 0.68; P = .04), and oxygen extraction ratio (rho = -0.71; P = .03). These relationships were absent in group noCy. CONCLUSIONS: The association of HSP-70i expression with improved right ventricular function and systemic perfusion suggests an important cardioprotective effect of HSP-70i in cyanotic tetralogy of Fallot.

Hypoxia upregulates GCM1 in human placenta explants.

Studies in mice have shown that a variety of genes, including GCM1, regulate the differentiation of trophoblast cells. GCM1 is also expressed in the human placenta. Placental GCM1 protein has been reported to be reduced in preeclampsia. In view of the close link between hypoxia, hypoxia-reoxygenation, preeclampsia, placental development and the reported reduction in GCM1, we hypothesised that GCM1 expression would be affected by hypoxia. The aim was to determine the effects of hypoxia on GCM1 expression in the human placenta. Two model systems were used; villous explants and cultured primary cytotrophoblast cells. GCM1 protein was detectable at low levels in explants maintained for 7 h in 8 or 20% O2. A striking increase in GCM1 was observed when villous explants were incubated for 1h in 1% O2 (p < 0.002). Incubation of explants for 1 h in 1% O(2) followed by re-oxygenation for 6 h in 8 or 20% O2 resulted in a decline in GCM1 protein. Expression of GCM1 was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in 8 or 20% O2 or reduced oxygen (1-2% O2) conditions. GCM1 protein was not detected in any of the experimental conditions used. This study has shown that acute hypoxia increases GCM-1 protein in villous explants. The experiments with purified trophoblast do not support a role for hypoxia increasing GCM-1 in these cells under the conditions used. The present findings are in keeping with the complex effects of oxygen depending on the conditions used. The hypoxic effects on GCM1 warrant further investigation as they may provide further information on the pathogenesis of preeclampsia.