Intraplaque hemorrhage.

Intraplaque hemorrhage is a common feature of atherosclerotic plaques and is considered one of the identifying features of complex lesions preceding acute ischemic events. The cause of intraplaque hemorrhage is most often secondary to rupture of neovessels, which have invaded the plaque. However, inflammation and metabolic factors such as diabetes may also precipitate hemorrhage from mature microvessels by damaging the endothelium. The mechanism by which hemorrhage destabilizes the plaque is in large part secondary to the action of hemoglobin released from red blood cells at the site of the hemorrhage. Hemoglobin is a potent pro-inflammatory agent by virtue of its ability to promote formation of ROS. The major defense mechanism against the toxic effects of extracorpuscular hemoglobin is the protein haptoglobin, which tightly binds to hemoglobin and prevents it from catalyzing oxidative reactions. There exists a common allelic polymorphism in the haptoglobin gene, which has recently been strongly associated with the risk of cardiovascular disease in multiple independent cohorts. The protein products of the two different haptoglobin alleles differ in their ability to serve as an antioxidant against hemoglobin and also to activate the CD163 receptor. This review presents a unifying hypothesis whereby the haptoglobin genotype is proposed to modulate the response to intraplaque hemorrhage and thereby play a fundamental role in determining the morphological and metabolic features of complex plaques preceding acute ischemic events.

Genetics of diabetic cardiovascular disease: identification of a major susceptibility gene.

Differential susceptibility to coronary artery disease in the diabetic patient cannot be entirely explained by conventional cardiac risk factors or by diabetes characteristics such as the degree of glycaemic control or diabetes duration. There is a growing awareness that there exist susceptibility genes for the development of diabetic vascular complications. In this review, I focus on one such genetic susceptibility factor encoded at the haptoglobin locus.

Structure-function analysis of the antioxidant properties of haptoglobin.

Haptoglobin serves as an antioxidant by virtue of its ability to prevent hemoglobin-driven 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 mini-haptoglobin described here, to avert the development of diabetic vascular complications.

Reciprocal regulation of expression of pore-forming KATP channel genes by hypoxia.

The ATP-sensitive potassium (KATP) channel is thought to play an important role in the protection of heart and brain against tissue hypoxia. The genetic regulation of the components of the channel by hypoxia has not been previously described. Here, we investigated the regulation of the two pore-forming channel proteins, Kir6.1 and Kir6.2, in response to hypoxia in vivo and in vitro. We find that these two structurally-related inwardly-rectifying potassium channel proteins are reciprocally regulated by hypoxia in vivo, with upregulation of Kir6.1 and down-regulation of Kir6.2, thereby resulting in a significant change in the composition of the channel complex in response to hypoxia. In vitro we describe neuronal and cardiac cell lines in which Kir6.1 is up-regulated by hypoxia, demonstrating that Kir6.1 is a hypoxia-inducible gene. We conclude that the heart and brain display genetic plasticity in response to hypoxic stress through specific genetic reprograming of cytoprotective channel genes.

Regulation of VEGF in diabetic patients with critical limb ischemia.

Diabetic patients are at a 10- to 20-fold increased risk for the development of critical limb ischemia. Vascular endothelial growth factor (VEGF) is critical for the development of collateral blood vessels, which can effectively bypass peripheral arterial occlusions. We therefore set out to determine if the regulation of VEGF in patients with peripheral vascular disease differs in diabetic and nondiabetic patients. Diabetic and nondiabetic patients with peripheral vascular disease were divided into those with or without critical limb ischemia as defined by clinical criteria (rest pain, nonhealing ulcer). Monocytes from peripheral blood were isolated from all patients and the hypoxic induction of VEGF was determined in vitro. In patients without diabetes, we found that there was no significant difference in the hypoxic induction of VEGF between patients with or without critical limb ischemia. However, in diabetic patients we found that patients with critical limb ischemia produced significantly more VEGF than patients without critical limb ischemia (6.3 +/- 1.3 vs. 2.1 +/- 0.3, p < 0.015). We conclude that diabetic patients with critical limb ischemia do not have an impairment in the ability to produce VEGF with hypoxia. Contrary to current dogma, treatment paradigms directed at increasing VEGF production in the diabetic patient with critical limb ischemia might not be beneficial.

Haptoglobin phenotype and diabetic nephropathy.

AIMS/HYPOTHESIS: To determine if the haptoglobin 2 allele is associated with an increased risk for the development of diabetic nephropathy. METHODS: This study included 110 consecutive normotensive subjects with Type I (insulin-dependent) diabetes mellitus and Type II (non-insulin-dependent) diabetes mellitus seen in two outpatient clinics in Israel. Diabetes duration was greater than 10 years for Type I diabetes and more than 5 years for Type II diabetic subjects. Microalbuminuria was defined as urinary protein excretion of 30 to 300 mg/24 h, and macroalbuminuria was defined as urinary protein excretion of greater than 300 mg/24 h. Serum was taken from subjects for haptoglobin typing by gel electrophoresis. RESULTS: Of the participating subjects 54 had Type I and 56 had Type II diabetes. None (0/18) of the subjects homozygous for the haptoglobin 1 allele (1-1) showed any sign of diabetic nephropathy, as compared with 34 % (19/55) of subjects homozygous for the haptoglobin 2 allele (2-2) and 27 % (10/37) of heterozygous subjects (2-1) (p < 0.04). Of the subjects 29 showed macroalbuminuria. The risk of developing macroalbuminuria was found to be greater in subjects with two haptoglobin 2 alleles (22 %) (12/55) as compared with one haptoglobin 2 allele (8 %) (3/37) or no haptoglobin 2 alleles (0%) (0/18) (p < 0.03). CONCLUSION/INTERPRETATION: By showing a graded risk relation to the number of haptoglobin 2 alleles in Type I and Type II diabetic subjects, these studies further support our hypothesis that the haptoglobin phenotype is a major susceptibility gene for the development of diabetic nephropathy.

Haptoglobin phenotype as a predictor of restenosis after percutaneous transluminal coronary angioplasty.

We have demonstrated that a genetic polymorphism in the antioxidant protein haptoglobin is important in determining which patients develop restenosis after percutaneous transluminal coronary angioplasty. Knowledge of the haptoglobin phenotype may be useful in the assessment and utilization of new therapies to reduce restenosis, particularly in patients who are homozygous for the haptoglobin 2 allele.

Hypoxic induction of vascular endothelial growth factor is markedly decreased in diabetic individuals who do not develop retinopathy.

OBJECTIVE: A small percentage of patients do not develop any evidence of diabetic retinopathy (DR) even after many years of diabetes. Vascular endothelial growth factor (VEGF) has been implicated in the development of DR. Therefore, we sought to determine if the regulation of VEGF differs in those patients who develop DR after many years of diabetes compared with those who do not develop DR. RESEARCH DESIGN AND METHODS: We performed standard 7-field stereoscopic fundus photography on 95 consecutive patients with type 1 and type 2 diabetes. Patients were categorized into 3 groups according to the presence or absence of DR and the duration of diabetes: group 1 was defined by presence of DR, group 2 was defined by absence of DR after >10 years duration of diabetes, and group 3 was defined by absence of DR with long-standing diabetes (> or =20 years for type 1 diabetes and > or =15 years for type 2 diabetes). Monocytes from 40 ml peripheral blood were isolated from all patients, and the hypoxic induction of VEGF was determined in vitro. RESULTS: We found no significant difference in the basal level of VEGF in patients with and without DR. However, we did find a markedly significant difference in the hypoxic induction of VEGF between patients from group 1 and group 3 (4.35+/-0.55 vs. 1.87+/-0.3, P<0.00013). CONCLUSIONS: This study suggests that 1 mechanism for the absence of DR in patients with long-standing diabetes is a decreased hypoxic induction of VEGF.

Bradycardia is associated with development of coronary collateral vessels in humans.

BACKGROUND: The development of mature coronary collateral vessels in patients with obstructive coronary artery disease (CAD) decreases the ischemic myocardial burden. Chronic bradycardia has been shown to stimulate formation of collateral vessels in experimental models. OBJECTIVE: To test our hypothesis that CAD patients with bradycardia would have better developed collateral circulation than would members of a control group. DESIGN: A retrospective study examining the relationship between bradycardia and the development of coronary collateral vessels in patients with obstructive CAD. METHODS: Admission electrocardiograms and rhythm tracings obtained during angiography of all patients presenting to the cardiac catheterization laboratory were screened from January to October 1997. Angiograms for patients with heart rates < or = 50 beats/min were reviewed. An equivalent number of consecutive patients with heart rates > or = 60 beats/min served as controls. Patients with acute myocardial infarction, with rhythms other than sinus, and without high grade obstructive CAD (< 70% stenosis) were excluded from the study. RESULTS: The study population consisted of 61 patients, 30 having heart rates < or = 50 beats/min (group A), and 31 controls with heart rates > or = 60 beats/min (group B). A significantly greater proportion of patients in group A than of matched controls was demonstrated to have developed collaterals (97 versus 55% in group B, P < 0.005). The mean collateral grades were 1.66 and 0.95 for subjects in groups A and B, respectively (P < 0.001). CAD patients with bradycardia are more likely (odds ratio 24, 95% confidence interval 5-146) to have angiographic coronary collaterals than are those with higher heart rates. CONCLUSION: Results of this study demonstrate that there is an association between bradycardia and growth of collateral vessels in patients with obstructive CAD. Bradycardic agents may be useful for promoting development of coronary collaterals in patients with atherosclerotic disease.

Complex contribution of the 3'-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR.

Cytokine stimulation of human DLD-1 cells resulted in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there was a basal transcription of the NOS II gene, which did not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR showed a significantly reduced luciferase activity. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to bind with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduced the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiated the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA.

Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability.

Stability of the vascular endothelial growth factor (VEGF) mRNA is tightly regulated through its 3'-untranslated region (3'-UTR). Here, we demonstrate that VEGF mRNA levels are increased by anisomycin, a strong activator of stress-activated protein kinases. Hence, VEGF mRNA induction is inhibited by SB202190, an inhibitor of JNK and p38/HOG kinase. Furthermore, VEGF mRNA expression is increased in cells that overexpress JNK and p38/HOG by an increase in its stability. We show by two different approaches that anisomycin exerts its effect on the VEGF mRNA 3'-UTR. First, by using an in vitro mRNA degradation assay, the half-life of the VEGF mRNA 3'-UTR region transcript was found to be increased when incubated with extracts from anisomycin-treated cells; and second, the 3'-UTR was also sufficient to confer mRNA instability to the Nhe3 (Na(+)/H(+) exchanger 3) heterologous reporter gene, and anisomycin treatment stabilized the chimeric mRNA (Nhe3 fused to the VEGF mRNA 3'-UTR). This chimeric mRNA is also more stable in cells overexpressing p38/HOG and JNK that have been stimulated by anisomycin. We show that such regulation is mediated through an AU-rich region of the 3'-UTR contained within a stable hairpin structure. By RNA electrophoretic mobility shift assays, we show that this region binds proteins specifically induced by anisomycin treatment. These findings clearly demonstrate a major role of stress-activated protein kinases in the post-transcriptional regulation of VEGF.

A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic states.

BACKGROUND: Angiogenesis, or new blood vessel formation, is the physiological adaptation of a tissue to hypoxia or ischemia. However, this compensatory response to hypoxic stress in vivo is often insufficient. In many of the conditions in which the angiogenic response to tissue hypoxia is insufficient, such as chronic critical limb ischemia or myocardial hibernation, the hypoxic stress is chronic and persistent, lasting for days or even months. Vascular endothelial growth factor (VEGF) has been demonstrated in vivo and in vitro to be the principal mediator of hypoxia-induced angiogenesis. We propose that the lack of compensatory angiogenesis in response to tissue hypoxia in many clinical syndromes characterized by chronic hypoxia is due to a failure to induce VEGF appropriately. METHODS AND RESULTS: Heart or liver cells were grown under conditions of chronic hypoxia, returned to a normoxic environment, and then rechallenged with hypoxia. We found that the hypoxic induction of VEGF mRNA was markedly blunted using this algorithm. Furthermore, transient transfection studies using the VEGF promoter containing an oxygen-responsive enhancer element failed to show induction in cells pretreated by subjection to chronic hypoxia. CONCLUSIONS: Hypoxic pretreatment results in a blunting of the ability of a cell to induce VEGF in response to subsequent episodes of hypoxia. This may provide a rationale for the inadequate amount of compensatory angiogenesis seen in many chronic ischemic disorders.

Interindividual heterogeneity in the hypoxic regulation of VEGF: significance for the development of the coronary artery collateral circulation.

BACKGROUND: The coronary artery collateral circulation may be beneficial in protecting against myocardial ischemia and necrosis. However, there is a tremendous interindividual variability in the degree of new collateral formation in patients with coronary artery disease. The basis for this interindividual heterogeneity is not understood. In this study we test the hypothesis that failure to generate collateral vessels is associated with a failure to appropriately induce with hypoxia or ischemia the angiogenic factor, vascular endothelial growth factor (VEGF). METHODS AND RESULTS: We correlated the VEGF response to hypoxia in the monocytes harvested from patients with coronary artery disease with the presence of collaterals visualized during routine angiography. We found that there was a highly significant difference in the hypoxic induction of VEGF in patients with no collaterals compared with patients with some collaterals (mean fold induction 1.9+/-0.2 versus 3.2+/-0.3, P<0.0001). After subjecting the data to ANCOVA, using as covariates a number of factors that might influence the amount of collateral formation (ie, age, sex, diabetes, smoking, hypercholesterolemia), patients with no collaterals still have a significantly lower hypoxic induction of VEGF than patients with collaterals. CONCLUSIONS: This study provides evidence in support of the hypothesis that the ability to respond to progressive coronary artery stenosis is strongly associated with the ability to induce VEGF in response to hypoxia. The observed interindividual heterogeneity in this response may be due to environmental, epigenetic, or genetic causes. This interindividual heterogeneity may also help to explain the variable angiogenic responses seen in other conditions such as diabetic retinopathy and solid tumors.

Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor.

Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out normal activities in a highly hypoxic environment. A key component of this adaptation is a higher capillary density in some Spalax tissues resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is critical for angiogenesis during development and in response to tissue ischemia. We demonstrate here that VEGF expression is markedly increased in those Spalax tissues with a higher capillary density relative to the normal laboratory rat Rattus norvegicus. Upregulation of VEGF thus appears to be an additional mechanism by which Spalax has adapted to its hypoxic environment.

The function of hypoxia-inducible factor 1 (HIF-1) is impaired in senescent mice.

Senescent organisms respond poorly to hypoxic stress. The transcription factor hypoxia-inducible factor 1 (HIF-1) plays a critical role in the coordinated genetic program that is induced in all tissues to adapt to hypoxic stress by binding to a specific DNA hypoxia-responsive recognition element (HRE). This study was designed to address whether aging is associated with an alteration in HIF-1 production and function. Young and old mice were exposed to hypoxia for various lengths of time. We found a severe impairment in the capacity of the old animals to form a HIF-1-HRE complex. This attenuation in the capacity to form HIF-1-HRE complexes in senescent tissues may explain the decreased ability of such tissues to respond to hypoxic stress.

Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is dramatically induced by hypoxia due in large part to an increase in the stability of its mRNA. Here we show that HuR binds with high affinity and specificity to the element that regulates VEGF mRNA stability by hypoxia. Inhibition of HuR expression abrogates the hypoxia-mediated increase in VEGF mRNA stability. Overexpression of HuR increases the stability of VEGF mRNA. However, this only occurs efficiently in hypoxic cells. We further show that the stabilization of VEGF mRNA can be recapitulated in vitro. Using an S-100 extract, we show that the addition of recombinant HuR stabilizes VEGF mRNA markedly. These data support the critical role of HuR in mediating the hypoxic stabilization of VEGF mRNA by hypoxia.

Hypoxic regulation of VEGF mRNA stability by RNA-binding proteins.

Vascular endothelial growth factor (VEGF), is a potent angiogenic factor whose expression is dramatically induced by hypoxia. We have previously demonstrated that the induction of VEGF by hypoxia is in large part the result of an increased stability of VEGF mRNA. The stabilization of VEGF mRNA by hypoxia is mediated by the binding of sequence-specific RNA-binding proteins. This review focuses on one such protein, HuR, an RNA-binding protein which we have recently shown is critical for the hypoxic stabilization of VEGF mRNA.

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascularization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/1 (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/l (383 ng/m1) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/l increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.