Vascular endothelial growth factor (VEGF), a potent angiogenic factor and endothelial cell-specific mitogen, is up-regulated by hypoxia. However, the mechanism(s) responsible for hypoxic induction of VEGF has not been clearly delineated. We report that the steady state VEGF mRNA levels are increased 12 +/- 0.6-fold, but the transcriptional rate for VEGF is increased only 3.1 +/- 0.6-fold by hypoxia in PC12 cells. In order to investigate cis-regulatory sequences which mediate this response to hypoxia, we cloned the rat genomic sequences encoding VEGF and identified a 28-base pair element in the 5' promoter that mediates hypoxia-inducible transcription in transient expression assays. This element has sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin 3' enhancer. Post-transcriptional mechanisms have also been suggested to play a role in the hypoxic induction of VEGF. Evidence is provided that a frequently used polyadenylation site is 1.9 kilobases downstream from the translation termination codon for rat VEGF. This site is 1.5 kilobases further downstream from the polyadenylation site previously reported for VEGF. This new finding reveals sequence motifs in the 3'-untranslated region that may mediate VEGF mRNA stability.

No abstract

The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 ؎ 6 min to 106 ؎ 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.Hypoxia has been shown to be an important stimulus for the new blood vessel formation seen in coronary artery disease (1), tumor angiogenesis (2), and diabetic neovascularization (3). VEGF, 1 also known as vascular permeability factor, is a potent angiogenic and endothelial cell-specific mitogen (4 -6), which is regulated by hypoxia in vitro (2,7,8) and in vivo (2, 3, 9 -11). The major control point for the hypoxic induction of the VEGF gene is the regulation of the steady-state level of mRNA (2, 8) which is determined by the relative rates of mRNA synthesis and decay.We have previously demonstrated that hypoxia induces VEGF steady-state mRNA 25.0 Ϯ 11.4 and 12.0 Ϯ 0.6 fold in rat primary cardiac myocytes (8) and rat pheochromocytoma PC12 cells (12), respectively. However, nuclear runoff transcription assays demonstrated that the transcription rate for VEGF was increased only 3.1 Ϯ 0.6-fold by hypoxia in the PC12 cells (12). Rat genomic sequences encoding VEGF were cloned and a 28-bp element in the 5Ј promoter was identified that mediates a significant portion of this hypoxia-inducible transcription in transient expression assays. This element was shown to have sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin (Epo) 3Ј enhancer (12). These studies demonstrated that, while increased transcription rate can account for a portion of the increase in the steady-state level of VEGF mRNA in the PC12 cells, it does not account for all of the increase and suggested that a post-transcriptional mechanism plays a significant role in the hypoxic induction of VEGF mRNA, as well.Post-transcriptional mechanisms of regulation have previously been suggested for Epo (13)(14)(15) and demonstrated for tyrosin...

Haptoglobin is an abundant hemoglobin-binding protein present in the plasma. The function of haptoglobin is primarily to determine the fate of hemoglobin released from red blood cells after either intravascular or extravascular hemolysis. There are two common alleles at the Hp genetic locus denoted 1 and 2. There are functional differences between the Hp 1 and Hp 2 protein products in protecting against hemoglobin-driven oxidative stress that appear to have important clinical significance. In particular, individuals with the Hp 2-2 genotype and diabetes mellitus appear to be at significantly higher risk of microvascular and macrovascular complications. A pharmacogenomic strategy of administering high dose antioxidants specifically to Hp 2-2 DM individuals may be clinically effective.

Haptoglobin serves as an antioxidant by virtue of its ability to prevent hemoglobindriven oxidative tissue damage. It was recently demonstrated that an allelic polymorphism in the haptoglobin gene is predictive of the risk for numerous microvascular and macrovascular diabetic complications. Because these complications are attributed in large part to an increase in oxidative stress, a study was conducted to determine whether the different protein products of the 2 haptoglobin alleles differed in the antioxidant protection they provided. A statistically significant difference was found in the antioxidant capacity of purified haptoglobin protein produced from the 2 different alleles, consistent with the hypothesis that differences in genetically determined antioxidant status may explain differential susceptibility to diabetic vascular complications. These differences may be amplified in the vessel wall because of differences in the sieving capacity of the haptoglobin types. Therefore, an attempt was made to identify the minimal haptoglobin sequences necessary to inhibit oxidation by hemoglobin in vitro, and 2 independent haptoglobin peptides that function in this fashion as efficiently as native haptoglobin were identified. Identification of the biochemical basis for differences among haptoglobin types may lead to the rational development of new pharmacologic agents, such as the minihaptoglobin described here, to avert the development of diabetic vascular complications. (Blood. 2001;98:3693-3698)

Abstract-A major function of haptoglobin (Hp) is to bind hemoglobin (Hb) to form a stable Hp-Hb complex and thereby prevent Hb-induced oxidative tissue damage. Clearance of the Hp-Hb complex can be mediated by the monocyte/macrophage scavenger receptor CD163. We recently demonstrated that diabetic individuals homozygous for the Hp 2 allele (Hp 2-2) were at 500% greater risk of cardiovascular disease (CVD) compared with diabetic individuals homozygous for the Hp 1 allele (Hp 1-1). No differences in risk by Hp type were seen in individuals without diabetes. To understand the relationship between the Hp polymorphism and diabetic CVD, we sought to identify differences in antioxidant and scavenging functions between the Hp types and to determine how these functions were modified in diabetes. The scavenging function of Hp was assessed using rhodamine-tagged and 125 I-Hp in cell lines stably transfected with CD163 and in macrophages expressing endogenous CD163. We found that the rate of clearance of Hp 1-1-Hb by CD163 is markedly greater than that of Hp 2-2-Hb. Diabetes is associated with an increase in the nonenzymatic glycosylation of serum proteins, including Hb. The antioxidant function of Hp was assessed with glycosylated and nonglycosylated Hb. We identified a severe impairment in the ability of Hp to prevent oxidation mediated by glycosylated Hb. We propose that the specific interaction between diabetes, CVD, and Hp genotype is the result of the heightened urgency of rapidly clearing glycosylated Hb-Hp complexes from the subendothelial space before they can oxidatively modify low-density lipoprotein to atherogenic oxidized low-density lipoprotein.

Hypoxia induces an increase in the stability of the mRNA encoding vascular endothelial growth factor (VEGF). We have previously demonstrated that a 500-base region of the 3-untranslated region of VEGF mRNA that is critical for stabilization of VEGF mRNA in an in vitro degradation assay forms a RNA-protein complex in a hypoxia-inducible fashion. We report here the identification of three adenylate-uridylate-rich RNA elements within this region that form an identical or closely related hypoxia-inducible RNA-protein complex. This complex is constitutively elevated in a tumor cell line lacking the wild type von Hippel-Lindau tumor suppressor gene and in which VEGF mRNA is constitutively stabilized. Furthermore, the glucose transporter-1 mRNA, which is also stabilized by hypoxia, forms a hypoxia-inducible RNA-protein complex with similar sequence and protein binding characteristics to that described for VEGF mRNA. Finally, RNA affinity purification and UV cross-linking were used to identify three proteins of 32, 28, and 17 kDa that are derived from this hypoxia-inducible RNA-protein complex.

Objective-Intraplaque hemorrhage increases the risk of plaque rupture and thrombosis. The release of hemoglobin (Hb) from extravasated erythrocytes at the site of hemorrhage leads to iron deposition, which may increase oxidation and inflammation in the atherosclerotic plaque. The haptoglobin (Hp) protein is critical for protection against Hb-induced injury. Two common alleles exist at the Hp locus and the Hp 2 allele has been associated with increased risk of myocardial infarction. We have demonstrated decreased anti-oxidative and anti-inflammatory activity for the Hp 2 protein. We tested the hypothesis that the Hp 2-2 genotype is associated with increased oxidative and macrophage accumulation in atherosclerotic plaques. Methods and Results-The murine Hp gene is a type 1 Hp allele. We created a murine type 2 Hp allele and targeted its insertion to the Hp locus by homologous recombination. Atherosclerotic plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 2-2 mice were associated with increased iron (Pϭ0.008), lipid peroxidation (4-hydroxynonenal and ceroid) and macrophage accumulation (Pϭ0.03) as compared with plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 1-1 mice. Conclusions-Increased iron, lipid peroxidation and macrophage accumulation in ApoEϪ/Ϫ Hp 2-2 plaques suggests that the Hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque hemorrhage. Key Words: atherosclerotic plaque Ⅲ hemoglobin Ⅲ inflammation Ⅲ iron Ⅲ macrophages T he major cause of acute coronary thrombosis is atherosclerotic plaque rupture and the precursor lesion has been termed the high-risk plaque. [1][2][3][4][5][6] Pathological features of highrisk plaques include a large lipid necrotic core, thin fibrous cap, inflammatory infiltrate, and intraplaque hemorrhage. [1][2][3][4][5][6] Extracorpuscular hemoglobin (Hb) released from red blood cells after intra-plaque hemorrhage represents a potent stimulus for inflammation within the plaque. It is becoming apparent that the frequency of microvascular hemorrhages has been severely underestimated and may occur in up to 40% of all advanced atherosclerotic plaques. 7 An important defense mechanism to counteract the effects of intra-plaque hemorrhage is mediated by haptoglobin (Hp), an abundant serum protein whose primary function is to bind to extracorpuscular Hb, thereby attenuating its oxidative and inflammatory potential. 8 Hp also promotes the clearance of extracorpuscular Hb via the CD163 scavenger receptor present on macrophages. 9 This scavenging pathway is the only mechanism that exists for removing free Hb released at extravascular sites, ie, at sites of hemorrhage within the atherosclerotic plaque.In humans there exist 2 classes of alleles for Hp, designated 1 and 2. The Hp polymorphism is a common polymorphism. In the western world, 16% of the population is Hp 1-1 (homozygous for the Hp 1 allele), 36% is Hp 2-2 (homozygous for the Hp 2 allele), and 48% is Hp 2-1 (heterozygote). 8 The Hp 2 allele is found only in humans. All other mammals, including higher primates have only th...