Ischemic preconditioning-mediated cardioprotection is disrupted in heterozygous Flt-1 (VEGFR-1) knockout mice

This study attempts to address an important clinical issue by identifying potential candidates of VEGF signaling through Flt-1 receptor that trigger angiogenic signal under ischemic stress. To determine the significance of VEGF-Flt-1 (VEGFR1) signaling in ischemic preconditioned (PC) myocardium, we used heterozygous Flt-1 knockout (KO) mice to dissect the pathway and identify candidate genes involved in VEGF signaling. DNA microarrays were employed to detect, characterize and distinguish altered myocardial gene expression by comparing between wild type (WT) CD-1 and heterozygous Flt-1 KO mice when exposed to ischemia (30 min) and reperfusion (2 h). Moreover, KO mice demonstrated reduced beneficial effects of PC when compared to the WT with PC. In the KO and WT mice, the % recovery of the left ventricular developed pressure and the maximum first derivative of the developed pressure after ischemia/reperfusion without PC were similar. However, when animals were subjected to PC, the left ventricular functional recovery throughout the reperfusion period was significantly lower in KO mice than in WT mice. These results indicate for the first time that in the heterozygous Flt-1 KO mice, PC is not as effective as that found in WT. This observation may be due to downregulation of several important genes such as growth-regulated oncogene 1 (Gro1), heat shock proteins (HSP), I kappa B kinase beta (IKK beta), colony-stimulating factor-1 (CSF-1) and annexin A7, suggesting the importance of VEGF-Flt-1 receptor signaling during PC.     

Hematological,Hepatic, and Retinal Phenotypes in Mice Deficient for Prolyl Hydroxylase Domain Proteins in the Liver

Prolyl hydroxylase domain (PHD) proteins catalyze oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor 1α and 2α, tagging them for pVHL-dependent polyubiquitination and proteasomal degradation. In this study, albumin Cre (Alb^Cre)–mediated, hepatocyte-specific triple disruption of Phd1, Phd2, and Phd3 (Phd(1/2/3)hKO) promoted liver erythropoietin (EPO) expression 1246-fold, whereas renal EPO was down-regulated to 6.7% of normal levels. In Phd(1/2/3)hKO mice, hematocrit levels reached 82.4%, accompanied by severe vascular malformation and steatosis in the liver. In mice double-deficient for hepatic PHD2 and PHD3 (Phd(2/3)hKO), liver EPO increase and renal EPO loss both occurred but were much less dramatic than in Phd(1/2/3)hKO mice. Hematocrit levels, vascular organization, and liver lipid contents all appeared normal in Phd(2/3)hKO mice. In a chronic renal failure model, Phd(2/3)hKO mice maintained normal hematocrit levels throughout the 8-week time course, whereas floxed controls developed severe anemia. Maintenance of normal hematocrit levels in Phd(2/3)hKO mice was accomplished by sensitized induction of liver EPO expression. Consistent with such a mechanism, liver HIF-2α accumulated to higher levels in Phd(2/3)hKO mice in response to conditions causing modest systemic hypoxia. Besides promoting erythropoiesis, EPO is also known to modulate retinal vascular integrity and neovascularization. In Phd(1/2/3)hKO mice, however, neonatal retinas remained sensitive to oxygen-induced retinopathy, suggesting that local EPO may be more important than hepatic and/or renal EPO in mediating protective effects in the retina.Prolyl hydroxylase domain (PHD) proteins PHD1, PHD2, and PHD3 use molecular oxygen as a substrate to hydroxylate specific prolyl residues in HIF-1α and HIF-2α,^1–3 tagging them for von Hippel–Lindau protein (pVHL)–dependent polyubiquitination and proteasomal degradation.⁴ PHD-regulated HIF-α stability is important for multiple processes, including angiogenesis,^5–7 erythropoiesis,^8–10 cardiomyocyte function,^11–13 cell survival,¹⁴ and metabolism.¹⁵ PHD1 and PHD2 also hydroxylate IKKβ, thus regulating the assembly of NF-κB and functions of monocytic cells and proangiogenic macrophages.^16–19 Besides PHDs, a transmembrane prolyl hydroxylase in the endoplasmic reticulum (P4H-TM) may also regulate HIF-α stability.^20,21In normal adults, renal interstitial cells are responsible for the bulk of plasma erythropoietin (EPO) and thus play a key role in regulating erythropoiesis and blood homeostasis. Loss or dysfunction of renal interstitial cells due to acute renal injury or chronic kidney disease can lead to EPO deficiency and severe anemia.²² Normal liver expresses EPO only at very low levels, but possesses latent capacity for EPO expression that can be reactivated by manipulations that lead to hepatic HIF-2α stabilization. For example, hepatocyte-specific Vhl knockout in mice resulted in the accumulation of HIF-1α and HIF-2α to high levels, and subsequent studies showed that HIF-2α was responsible for elevated liver EPO expression and polycythemia.^23,24 Other studies have also demonstrated critical roles of HIF-2α in regulating EPO expression.^25,26Our research group has previously shown that germline Phd1 and Phd3 double knockout leads to increased liver EPO expression.⁸ In a more recent study, Minamishima and Kaelin²⁷ showed that more dramatic liver EPO up-regulation can be induced by triple Phd knockout, with Phd1 and Phd3 knockout being germline null mutations and Phd2 knockout induced in a hepatocyte-restricted manner. These studies raised the possibility that the liver may be exploited as an alternative source for endogenous EPO production in case of renal failure. Indeed, siRNA-mediated knockdown of hepatic PHD rescued erythropoiesis in mice subjected to 5/6 nephrectomy.²⁸ Although these findings are highly encouraging, it is not known how the liver itself is affected by PHD deficiency.In the present study, we examined hematological effects of hepatic PHD deficiency in an established chronic renal failure model,^29–32 and compared blood, vascular, and lipid phenotypes associated with the disruption of different combinations of PHD isoforms in the liver. Hepatic triple deficiency of all three isoforms caused multiple abnormalities, including severe erythrocytosis, vascular malformation, and massive lipid accumulation in the liver. By contrast, mice double-deficient for hepatic PHD2 and PHD3 did not exhibit any of these defects, but yet gained the ability to maintain normal hematocrit (Hct) levels in a chronic renal failure model. These data provide the proof of principle that selective combinations of hepatic PHD isoforms could offer suitable therapeutic targets for maintaining normal blood homeostasis without accompanying vascular malformation or liver steatosis.