Relationship Between Time-Varying Achieved High-Density Lipoprotein Cholesterol and Risk of Coronary Events Depends on Haptoglobin Phenotype Within the ACCORD Lipid Study.

Background The Hp (haptoglobin)2-2 phenotype (~40% of people) is associated with dysfunctional high-density lipoprotein (HDL) that is heavily oxidized in hyperglycemia, which may explain why raising HDL-cholesterol (HDL-C) does not reliably prevent coronary artery disease (CAD) in diabetes. Methods and Results In this observational study using longitudinal data from the ACCORD (Action to Control Cardiovascular Risk in Diabetes) lipid trial, time-varying (achieved) HDL-C updated at 4, 8, and 12 months, and annually thereafter over a mean of 4.7 years, was analyzed in relation to risk of CAD and secondary outcomes using Cox proportional hazards regression with time-varying covariables among participants with (n=1781) and without (n=3191) the Hp2-2 phenotype. HDL-C did not differ between the phenotypes throughout the study. Having low HDL-C (<40 mg/dL for male participants and <50 mg/dL for female participants) was associated with a greater risk of CAD compared with non-low HDL-C among participants with the non-Hp2-2 phenotype (hazard ratio [HR], 1.48 [95% CI, 1.18-1.87]) but not among the Hp2-2 phenotype (HR, 0.97 [95% CI, 0.70-1.35]; P interaction=0.03). Similarly, an inverse relationship was observed between HDL-C quintiles and CAD risk among participants without the Hp2-2 phenotype, whereas no significant inverse relationship was observed among participants with the Hp2-2 phenotype (P interaction=0.38). Among the Hp2-2 phenotype group, having low HDL-C was associated with higher risk of CVD mortality (HR, 2.09 [95% CI, 1.05-4.13]), and compared with the lowest HDL-C quintile, higher quintiles were associated with lower risk of CVD mortality and congestive heart failure. Conclusions Hp phenotype modified the association between HDL-C and risk of CAD in the ACCORD lipid study, suggesting that HDL dysfunction in the Hp2-2 phenotype may hinder CAD-protective properties of HDL-C.

The Relationship Between Time-Varying Achieved HbA1c and Risk of Coronary Events Depends on Haptoglobin Phenotype Among White and Black ACCORD Participants.

OBJECTIVE: Intensive glycemic therapy reduced coronary artery disease (CAD) events among White participants in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study with the haptoglobin (Hp)2-2 phenotype, while participants without the Hp2-2 phenotype had no CAD benefit. The association between achieved glycated hemoglobin (HbA1c) and CAD for each Hp phenotype remains unknown. RESEARCH DESIGN AND METHODS: Achieved HbA1c was similar in each phenotype throughout the study. Prospectively collected HbA1c data (categorized as <6.0%, 6.0-6.5%, 6.6-6.9%, or ≥8.0% compared with 7.0-7.9%) from the ACCORD study, updated every 4 months over a median of 4.7 years, were analyzed in relation to CAD in the Hp2-2 (n = 3,322) and non-Hp2-2 (n = 5,949) phenotypes separately overall, and within White (63%, 37% Hp2-2) and Black (19%, 26% Hp2-2) participants using Cox proportional hazards regression with time-varying covariables. RESULTS: Compared with HbA1c of 7.0-7.9%, having HbA1c ≥8.0% was associated with CAD risk among White (adjusted HR [aHR] 1.43, 95% CI 1.03-1.98) and Black (2.86, 1.09-7.51) participants with the Hp2-2 phenotype, but not when all Hp2-2 participants were combined overall (1.30, 0.99-1.70), and not among participants without the Hp2-2 phenotype. HbA1c <7.0% was not associated with a lower risk of CAD for any Hp phenotype. CONCLUSIONS: Achieving HbA1c >8.0% compared with 7.0-7.9% was consistently associated with incident CAD risk among White and Black ACCORD participants with the Hp2-2 phenotype, while no association was observed among participants without the Hp2-2 phenotype. We found no evidence that HbA1c concentration <7.0% prevents CAD in either Hp phenotype group.

Molecular Insights into IQSEC2 Disease.

Recent insights into IQSEC2 disease are summarized in this review as follows: (1) Exome sequencing of IQSEC2 patient DNA has led to the identification of numerous missense mutations that delineate at least six and possibly seven essential functional domains present in the IQSEC2 gene. (2) Experiments using IQSEC2 transgenic and knockout (KO) mouse models have recapitulated the presence of autistic-like behavior and epileptic seizures in affected animals; however, seizure severity and etiology appear to vary considerably between models. (3) Studies in IQSEC2 KO mice reveal that IQSEC2 is involved in inhibitory as well as stimulatory neurotransmission. The overall picture appears to be that mutated or absent IQSEC2 arrests neuronal development, resulting in immature neuronal networks. Subsequent maturation is aberrant, leading to increased inhibition and reduced neuronal transmission. (4) The levels of Arf6-GTP remain constitutively high in IQSEC2 knockout mice despite the absence of IQSEC2 protein, indicating impaired regulation of the Arf6 guanine nucleotide exchange cycle. (5) A new therapy that has been shown to reduce the seizure burden for the IQSEC2 A350V mutation is heat treatment. Induction of the heat shock response may be responsible for this therapeutic effect.

Haptoglobin Phenotype Modifies the Influence of Intensive Glycemic Control on Cardiovascular Outcomes.

BACKGROUND: Whereas there exists a direct relationship between glycated hemoglobin and cardiovascular disease (CVD), clinical trials targeting glycated hemoglobin to near-normal levels using intensive therapy have failed to prevent CVD and have even increased mortality, making clinical decision making difficult. A common polymorphism at the haptoglobin (Hp) genetic locus is associated with CVD, especially coronary heart disease, in the setting of hyperglycemia. OBJECTIVES: This study sought to determine whether the treatment difference of intensive versus standard glucose-lowering therapy on risk of CVD events in the ACCORD (Action to Control Cardiovascular Risk in Diabetes) study depended on Hp phenotype. METHODS: Hp phenotype was measured within 5,806 non-Hispanic white ACCORD participants using a validated assay. Adjusted hazard ratios (aHR) with 95% confidence intervals (CI) estimated from stratified Cox regression models were used to quantify the association between intensive therapy and incident CVD for the 2 different Hp phenotype groups (Hp2-2, Hp1 carriers). RESULTS: Compared with standard therapy, intensive therapy was associated with a lower risk of incident coronary heart disease among participants with the Hp2-2 phenotype (n = 2,133; aHR: 0.71; 95% CI: 0.55 to 0.91; p = 0.006), but not among the other 2 phenotypes (Hp1 allele carriers) (n = 3,673; aHR: 0.95; 95% CI: 0.79 to 1.13; p = 0.550). The same pattern was observed for CVD. Conversely, intensive therapy was associated with an increased risk of fatal CVD (aHR: 1.50; 95% CI: 1.00 to 2.25; p = 0.049) and total mortality (aHR: 1.40; 95% CI: 1.08 to 1.81; p = 0.011) among the Hp1 carriers, whereas this risk was not increased in the Hp2-2 phenotype (fatal CVD: aHR: 1.02; 95% CI: 0.59 to 1.77; p = 0.931; total mortality: aHR: 0.98; 95% CI: 0.68 to 1.41; p = 0.908). CONCLUSIONS: Intensive glucose-lowering therapy was effective at preventing incident coronary heart disease and CVD events in ACCORD study participants with the Hp2-2 phenotype but not in Hp1 carriers, who had increased mortality risk from intensive therapy.

IQSEC2-Associated Intellectual Disability and Autism.

Mutations in IQSEC2 cause intellectual disability (ID), which is often accompanied by seizures and autism. A number of studies have shown that IQSEC2 is an abundant protein in excitatory synapses and plays an important role in neuronal development as well as synaptic plasticity. Here, we review neuronal IQSEC2 signaling with emphasis on those aspects likely to be involved in autism. IQSEC2 is normally bound to N-methyl-D-aspartate (NMDA)-type glutamate receptors via post synaptic density protein 95 (PSD-95). Activation of NMDA receptors results in calcium ion influx and binding to calmodulin present on the IQSEC2 IQ domain. Calcium/calmodulin induces a conformational change in IQSEC2 leading to activation of the SEC7 catalytic domain. GTP is exchanged for GDP on ADP ribosylation factor 6 (ARF6). Activated ARF6 promotes downregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors through a c-jun N terminal kinase (JNK)-mediated pathway. NMDA receptors, AMPA receptors, and PSD-95 are all known to be adversely affected in autism. An IQSEC2 transgenic mouse carrying a constitutively active mutation (A350V) shows autistic features and reduced levels of surface AMPA receptor subunit GluA2. Sec7 activity and AMPA receptor recycling are presented as two targets, which may respond to drug treatment in IQSEC2-associated ID and autism.

COPD Exacerbator Phenotype is Inversely Associated with Current Smoking But Not with Haptoglobin Phenotype.

BACKGROUND: Frequent chronic obstructive pulmonary disease (COPD) exacerbators are at a higher risk of adverse health outcomes when compared to infrequent exacerbators. A COPD frequent exacerbator phenotype and its definition has been reported. Haptoglobin (Hp) polymorphism has been associated with differing clinical outcomes in cardiovascular and renal disease. The Hp 2-2 phenotype has been found to have bacteriostatic properties, while the Hp 1-1 phenotype was found to be associated with infections. OBJECTIVES: To determine the correlation in haptoglobin phenotypes and the frequent exacerbator status compared to COPD non-exacerbators. METHODS: Inclusion criteria included previous diagnosis of COPD and presence of at least two documented exacerbations of COPD in the previous 12 months (frequent exacerbator group) or absence of such exacerbations in the previous 24 months (non-exacerbator group). Descriptive data was analyzed using Fisher's exact test and the nonparametric Kruskal-Wallis test. Multivariate logistic regression analysis was performed. RESULTS: The multivariate logistic regression yielded a model in which haptoglobin phenotype did not have a statistically significant association with frequent exacerbator status. Smoking status was found to be negatively related with the frequent exacerbator status (odds ratio [OR] 0.240, 95% confidence interval (95%CI) 0.068-0.843, P = 0.03). Number of pack-years was negatively related to being a frequent exacerbator (OR 0.979, 95%CI 0.962-0.996, P = 0.02). CONCLUSIONS: We found no relationship between haptoglobin polymorphism and frequent exacerbator status. However, frequent exacerbator status had a statistically significant association with COPD Assessment Test scores and pack-years and a negative correlation with current smoking status.

Association of the Haptoglobin Gene Polymorphism With Cognitive Function and Decline in Elderly African American Adults With Type 2 Diabetes: Findings From the Action to Control Cardiovascular Risk in Diabetes-Memory in Diabetes (ACCORD-MIND) Study.

Importance: African American individuals have higher dementia risk than individuals of white race/ethnicity. They also have higher rates of type 2 diabetes, which may contribute to this elevated risk. This study examined the association of the following 2 classes of alleles at the haptoglobin (Hp) locus that are associated with poor cognition, cardiovascular disease, and mortality: Hp 1-1 (associated with poor cognition and cerebrovascular disease) and Hp 2-1 and Hp 2-2 (associated with greater risk of myocardial infarction and mortality). An additional polymorphism in the promoter region of the Hp 2 allele, restricted to individuals of African descent, yields a fourth genotype, Hp 2-1m. African American adults have a higher prevalence of Hp 1-1 (approximately 30%) compared with individuals of white race/ethnicity (approximately 14%), but the potential role of the Hp genotype in cognition among elderly African American individuals with type 2 diabetes is unknown. Objective: To assess the association of the Hp genotypes with cognitive function and decline in elderly African American adults with type 2 diabetes. Design, Setting, and Participants: This cohort study used publicly available data and specimens from the Action to Control Cardiovascular Risk in Diabetes-Memory in Diabetes (ACCORD-MIND) study to investigate the association of the Hp genotypes with cognitive function and decline in 466 elderly African American participants with type 2 diabetes. The hypothesis was that the Hp 1-1 genotype compared with the other genotypes would be associated with more cognitive impairment and faster cognitive decline in elderly African American adults with type 2 diabetes. The initial ACCORD trial was performed from October 28, 1999, to September 15, 2014. This was a multicenter clinical study performed in an academic setting. Exposures: The Hp genotypes were determined from serum samples by polyacrylamide gel electrophoresis and by enzyme-linked immunosorbent assay. Main Outcomes and Measures: The Mini-Mental State Examination (MMSE) was used to measure cognitive function and change after 40 months. The MMSE score ranges from 0 to 30 points; higher scores represent better cognition. Associations were examined with analysis of covariance and linear regression, adjusting for age, sex, education, baseline glycated hemoglobin level, systolic blood pressure, diastolic blood pressure, cholesterol level, creatinine level, and treatment arm (intensive vs standard). The cognitive change model adjusted also for the baseline MMSE score. Results: Among 466 African American study participants (mean [SD] age, 62.3 [5.7] years), 64.8% were women, and the genotype prevalences were 29.4% (n = 137) for Hp 1-1, 36.1% (n = 168) for Hp 2-1, 10.9% (n = 51) for Hp 2-1m, and 23.6% (n = 110) for Hp 2-2. The groups differed in their baseline MMSE scores (P = .006): Hp 1-1 had the lowest MMSE score (mean [SE], 25.68 [0.23]), and Hp 2-1m had the highest MMSE score (mean [SE], 27.15 [0.36]). Using the least squares method, the 40-month decline was significant for Hp 1-1 (mean [SE], -0.41 [0.19]; P = .04) and for Hp 2-2 (mean [SE], -0.68 [0.21]; P = .001). However, the overall comparison across the 4 groups did not reach statistical significance for the fully adjusted model. The interaction of age with the Hp 1-1 genotype on MMSE score decline estimate per year change was significant (mean [SE], -0.87 [0.37]; P = .005), whereas it was not significant for Hp 2-1 (mean [SE], 0.06 [0.37]; P = .85), Hp 2-1m (mean [SE], -0.06 [0.51]; P = .89), and Hp 2-2 (mean [SE], -0.44 [0.41]; P = .29), indicating that cognitive decline in Hp 1-1 carriers was accentuated in older ages, whereas it was not significant for the other Hp genotypes. Conclusions and Relevance: In this study, the Hp 1-1 genotype, which is 2-fold (approximately 30%) more prevalent among African American individuals than among individuals of white race/ethnicity, was associated with poorer cognitive function and greater cognitive decline than the other Hp genotypes. The Hp gene polymorphism may explain the elevated dementia risk in African American adults. The neuropathological substrates and mechanisms for these associations merit further investigation.

An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype.

BACKGROUND: Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. METHODS: A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. RESULTS: Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. CONCLUSIONS: The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.

Haptoglobin genotype predicts development of coronary artery calcification in a prospective cohort of patients with type 1 diabetes.

BACKGROUND: Coronary artery disease has been linked with genotypes for haptoglobin (Hp) which modulates extracorpuscular hemoglobin. We hypothesized that the Hp genotype would predict progression of coronary artery calcification (CAC), a marker of subclinical atherosclerosis. METHODS: CAC was measured three times in six years among 436 subjects with type 1 diabetes and 526 control subjects participating in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. Hp typing was performed on plasma samples by polyacrylamide gel electrophoresis. RESULTS: The Hp 2-2 genotype predicted development of significant CAC only in subjects with diabetes who were free of CAC at baseline (OR: 1.95, 95% CI: 1.07-3.56, p = 0.03), compared to those without the Hp 2-2 genotype, controlling for age, sex, blood pressure and HDL-cholesterol. Hp 2 appeared to have an allele-dose effect on development of CAC. Hp genotype did not predict CAC progression in individuals without diabetes. CONCLUSIONS: Hp genotype may aid prediction of accelerated coronary atherosclerosis in subjects with type 1 diabetes.

Vitamin E therapy results in a reduction in HDL function in individuals with diabetes and the haptoglobin 2-1 genotype.

OBJECTIVE: Vitamin E provides cardiovascular protection to individuals with diabetes and the haptoglobin 2-2 genotype but appears to increase cardiovascular risk in individuals with diabetes and the haptoglobin 2-1 genotype. We have previously demonstrated that the haptoglobin protein is associated with HDL and that HDL function and its oxidative modification are haptoglobin genotype dependent. We set out to test the hypothesis that the pharmacogenetic interaction between the haptoglobin genotype on cardiovascular risk might be secondary to a parallel interaction between the haptoglobin genotype and vitamin E on HDL function. RESEARCH DESIGN AND METHODS: Fifty-nine individuals with diabetes and the haptoglobin 2-1 or 2-2 genotypes were studied in a double-blind placebo controlled crossover design. Participants were treated with either vitamin E (400IU) or placebo for 3 months and crossed over for an equivalent duration. Serum was collected at baseline and after the completion of each treatment. HDL functionality as well as HDL associated markers of oxidation and inflammation were measured after each interval in HDL purified from the cohort. RESULTS: Compared to placebo, vitamin E significantly increased HDL function in haptoglobin 2-2 but significantly decreased HDL function in haptoglobin 2-1. This pharmacogenetic interaction was paralleled by similar non-significant trends in HDL associated lipid peroxides, glutathione peroxidase, and inflammatory cargo. CONCLUSION: There exists a pharmacogenetic interaction between the haptoglobin genotype and vitamin E on HDL function (clinicaltrials.gov NCT01113671).

Haptoglobin: basic and clinical aspects.

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.

Increased blood vessel density provides the mole rat physiological tolerance to its hypoxic subterranean habitat.

The blind subterranean mole rat superspecies Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out intensive activities in its highly hypoxic underground sealed burrows. A key component of this adaptation is a higher capillary density in some Spalax tissues, primarily in muscles used in digging and in other energetic activities, 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 is found in response to tissue ischemia. We demonstrate here that due to physiological differences, the Spalax muscle regulatory mechanism for VEGF is different than in Rattus muscle. In vivo, the constitutive level of the VEGF mRNA and the mRNA levels of its transcriptional regulator HIF-1alpha and its mRNA stabilizer HuR are significantly higher in Spalax muscle than in Rattus muscle. Furthermore, as opposed to Rattus, the mRNA levels of HIF-1alpha, HuR, VEGF, as well as that of LDH-A, the enzyme that catalyzes the production of lactate, an accepted marker of anaerobic metabolism, are not increased in Spalax after hypoxia. However, ex vivo, when oxygenation by blood vessels is no longer relevant, the expression pattern of all these genes is similar in the two rodents under both normoxic and hypoxic conditions. Our studies provide evidence that the highly vascularized muscle in Spalax, the most energy consuming tissue during digging, is resistant to the effects of oxygen deprivation. The significance of these results with respect to ischemic vascular disease is abundantly clear.

Vascular endothelial growth factor (VEGF) fails to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model.

BACKGROUND: Angiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model. METHODS: Diabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle. RESULTS: VEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF. CONCLUSIONS: Angiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

Genetically determined heterogeneity in hemoglobin scavenging and susceptibility to diabetic cardiovascular disease.

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 125I-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.

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF.

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF +/- internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 microg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF-IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF-IRES (-) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.