Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.
We employed Cre/loxP technology to generate mPDK1 ±/± mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6-phosphofructo-2-kinase and production of fructose 2,6-bisphosphate, in the hearts of mPDK1 ±/± mice, consistent with PDK1 mediating these processes. All mPDK1 ±/± mice died suddenly between 5 and 11 weeks of age. The mPDK1 ±/± animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1 ±/± muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1 ±/± mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single-cell assay we found that cardiomyocytes from mPDK1 ±/± mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a de®ciency of the PDK1 pathway might contribute to development of cardiac disease in humans. Keywords: cardiac muscle/heart failure/hypoxia/PDK1/ PI 3-kinase/PKB/Akt IntroductionHormones and growth factors trigger the activation of members of a group of protein kinases including protein kinase B (PKB) and p70 ribosomal S6K (S6K), which belong to the AGC family of protein kinases (Brazil and Hemmings, 2001;Lawlor and Alessi, 2001; Newton, 2002). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating these AGC kinase members by phosphorylating these enzymes at their activation loop (Toker and Newton, 2000;Alessi, 2001). Much research has shown that the PDK1/AGC kinasesignalling pathway regulates diverse cellular processes, such as those relevant to cell survival, proliferation and metabolic responses to insulin. Misregulation of AGC kinase members is thought to contribute to many diseases. For example, hyperactivation of this pathway is implicated in inducing cardiac hypertrophy (Sugden, 2001) and promoting the survival and proliferation of a signi®cant number of cancers (Simpson and Parsons, 2001). A de®ciency in the activation of AGC kinases may be a primary cause of the insulin-resistant form of diabetes (Saltiel and Kahn, 2001), as well as neuronal cell death following a stroke (Wick et al., 2002).The activation of PKB and S6K isoforms by insulin and growth factors, as well as being dependent on PDK1, requires the prior activation of the phosphoinositide 3-kinase (PI 3-kinase) (Vanhaesebroeck et al., 2001). This produces the second messenger, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which binds to the pleckstrin homology domains of PKB and PDK1, recruiting these enzymes to the plasma membrane where PKB is activated by phosphorylation of its activation-loop residue (Thr308 in PKBa) by PDK1 (Brazil and Hemmings, 2001;Scheid and Woodgett, 2001). PtdIns(3,4,5)P 3 also stimulates the phosphorylation of PKB at its hydrophobic motif res...
Angiogenesis restores blood flow to healing tissues, a process that is inhibited by high doses of glucocorticoids. However, the role of endogenous glucocorticoids and the potential for antiglucocorticoid therapy to enhance angiogenesis is unknown. Using in vitro and in vivo models of angiogenesis in mice, we examined effects of (i) endogenous glucocorticoids, (ii) blocking endogenous glucocorticoid action with the glucocorticoid receptor antagonist RU38486, and (iii) abolishing local regeneration of glucocorticoids by the enzyme 11-hydroxysteroid dehydrogenase type 1 (11HSD1). Glucocorticoids, administered at physiological concentrations, inhibited angiogenesis in an in vitro aortic ring model and in vivo in polyurethane sponges implanted s.c. RU38486-enhanced angiogenesis in s.c. sponges, in healing surgical wounds, and in the myocardium of mice 7 days after myocardial infarction induced by coronary artery ligation. 11HSD1 knockout mice showed enhanced angiogenesis in vitro and in vivo within sponges, wounds, and infarcted myocardium. Endogenous glucocorticoids, including those generated locally by 11HSD1, exert tonic inhibition of angiogenesis. Inhibition of 11HSD1 in liver and adipose has been advocated to reduce cardiovascular risk in the metabolic syndrome: these data suggest that 11HSD1 inhibition offers a previously uncharacterized therapeutic approach to improve healing of ischemic or injured tissue. myocardial infarction ͉ wound healing
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