HIF hydroxylase pathways in cardiovascular physiology and medicine.

Hypoxia inducible factors (HIFs) are α/ß heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.

Aromatase activity after a short-course of letrozole administration in adult men at sea level and at high altitude (with or without excessive erythrocytosis).

Men living at high altitudes in Peru compared to sea level counterparts have erythrocytosis (hemoglobin 16-21 g/dl) or excessive erythrocytosis (hemoglobin>21 g/dl). High testosterone (T) levels in men at high altitude (HA) were associated with excessive erythrocytosis. High androgen levels could be due to a low aromatase activity or to an elevated rate of conversion from precursors to testosterone. The aim of this study was to evaluate aromatase activity and rate of conversion from precursors to testosterone before and after administration of the aromatase enzyme inhibitor letrozole (5 mg/day) for a 5-day period to men at HA and at sea level (SL). The response to short term aromatase inhibition was assessed in 30 adult men living at sea level, 31 native men at HA with erythrocytosis (Hb 16-21 g/dl), and 35 men at HA with excessive erythrocytosis (Hb>21 g/dl). Serum hormone levels, estradiol/testosterone, testosterone/androstenedione, and testosterone/dehydroepiandrosterone sulfate (DHEAS) ratios were measured. Men with erythrocytosis had lower basal serum T/androstenedione ratios than men with excessive erythrocytosis at HA and men at sea level. Men at HA with excessive erythrocytosis had higher T/DHEAS ratios than men with erythrocytosis and than those at sea level before and after letrozole administration. After letrozole administration, both groups of men at high altitude (with erythrocytosis or with excessive erythrocytosis) showed lower aromatase activities than those at sea level. In conclusion, higher serum testosterone levels in men with excessive erythrocytosis were associated with an increased rate of conversion from DHEAS to testosterone rather than to a lower aromatase activity.

A feedback loop involving the Phd3 prolyl hydroxylase tunes the mammalian hypoxic response in vivo.

Hypoxia-inducible factor (HIF), consisting of a labile alpha subunit and a stable beta subunit, is a master regulator of hypoxia-responsive mRNAs. HIF alpha undergoes oxygen-dependent prolyl hydroxylation, which marks it for polyubiquitination by a complex containing the von Hippel-Lindau protein (pVHL). Among the three Phd family members, Phd2 appears to be the primary HIF prolyl hydroxylase. Phd3 is induced by HIF and, based on findings from in vitro studies, may participate in a HIF-regulatory feedback loop. Here, we report that Phd3 loss exacerbates the HIF activation, hepatic steatosis, dilated cardiomyopathy, and premature mortality observed in mice lacking Phd2 alone and produces a closer phenocopy of the changes seen in mice lacking pVHL than the loss of Phd2 alone. Importantly, the degree to which Phd3 can compensate for Phd2 loss and the degree to which the combined loss of Phd2 and Phd3 resembles pVHL loss appear to differ for different HIF-responsive genes and in different tissues. These findings highlight that the responses of different HIF target genes to changes in prolyl hydroxylase activity differ, quantitatively and qualitatively, in vivo and have implications for the development of paralog-specific prolyl hydroxylase inhibitors as therapeutic agents.

Detection of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia vera.

To further characterize JAK2 exon 12 mutations, we performed molecular screening in 409 patients with polycythemia vera or unclear erythrocytosis with unmutated JAK2V617. The frequency of JAK2exon12 mutations was 10/63 (15.9%) in PV but only 5/346 (1.4%) in the erythrocytosis cases. Nine different mutations including four new types (D544-L545del, H538DK539LI540S, H538-K539del, V536-F547dup) were detected. In 2 cases we found evidence for the presence of cells homozygous for mutated JAK2exon12. As this was the case in only 2/15 cases with JAK2exon12 mutations (13%) homozygosity seemed to be less frequent than in V617F-mutated polycythemia vera (69%) (p<0.001). There were more females than males in the group of patients with a JAK2exon12 mutation (10 vs. 5) compared to the group with wildtype JAK2 (132 vs. 262; p=0.012). Median age of onset was lower than in the V617Fmut controls (58.5 vs. 67.8 years, p<0.001). In conclusion, JAK2 exon 12 mutation analysis contributes to diagnostics in polycythemia vera or erythrocytosis.

JAK2 V617F mutation is rare in idiopathic erythrocytosis: a difference from polycythemia vera.

A single mutation 1849G>T in the JAK2 gene (V617F) has recently been described in classical myeloproliferative disorders (MPD). To investigate the incidence and clinical significance of the JAK2 mutation, we performed allele-specific polymerase chain reaction (PCR) and enzyme-based assessment in 11 idiopathic erythrocytosis (IE) and 15 polycythemia vera (PV) patients. Aberrant bands indicating the V617F mutation were detected in only one of 11 patients with IE, whereas all of the 15 patients with PV showed the JAK2 mutation. Sequence analysis was subsequently performed in the IE patient showing aberrant bands on allele-specific PCR, and a nucleotide change corresponding to the V617F mutation was detected in four of 29 clones tested. This patient might have progressed to PV according to the new WHO diagnostic criteria proposed in 2007, since a gradual increase in platelet counts was observed 4 years after the time of diagnosis. A further longitudinal study monitoring V617F positive-cells will clarify the process of progression from IE to PV in such a patient.

Somatic inactivation of the PHD2 prolyl hydroxylase causes polycythemia and congestive heart failure.

Pharmacologic activation of the heterodimeric HIF transcription factor appears promising as a strategy to treat diseases, such as anemia, myocardial infarction, and stroke, in which tissue hypoxia is a prominent feature. HIF accumulation is normally linked to oxygen availability because an oxygen-dependent posttranslational modification (prolyl hydroxylation) marks the HIFalpha subunit for polyubiquitination and destruction. Three enzymes (PHD1, PHD2, and PHD3) capable of catalyzing this reaction have been identified, although PHD2 (also called Egln1) appears to be the primary HIF prolyl hydroxylase in cell culture experiments. We found that conditional inactivation of PHD2 in mice is sufficient to activate a subset of HIF target genes, including erythropoietin, leading to striking increases in red blood cell production. Mice lacking PHD2 exhibit premature mortality associated with marked venous congestion and dilated cardiomyopathy. The latter is likely the result of hyperviscosity syndrome and volume overload, although a direct effect of chronic, high-level HIF stimulation on cardiac myocytes cannot be excluded.

JAK2 exon 12 mutations in polycythemia vera or idiopathic erythrocytosis.

JAK2 exon 12 mutations were detected in 4 out of 20 polycythemia vera and idiopathic erythrocytosis V617F-negative patients and were only present in the myeloid lineage. Initial hematologic data of these patients differ from those of V617F-positive patients, but there is no difference in thrombotic development and myelofibrotic transformation.

Usefulness of JAK2V617F mutation in distinguishing idiopathic erythrocytosis from polycythemia vera.

Idiopathic erythrocytosis (IE) is a primary erythrocytosis not fulfilling the criteria for polycythemia vera (PV) diagnosis. In order to verify the relationship between IE and PV, we screened JAK2V617F mutation in a consecutive series of 11 IE and, for comparison, in 15 PV. JAK2V617F mutation was screened by both cDNA sequencing and mutation specific PCR in both peripheral blood and bone marrow samples. All 11 IE tested negative for JAK2V617F mutation, which, conversely, occurred in 11/15 (73.3%) PV. Our results demonstrate that JAK2V617F is absent in IE and may represent a useful molecular marker for distinguishing IE from PV.

The incidence of the JAK2 V617F mutation in patients with idiopathic erythrocytosis.

Sixty-three patients with erythrocytosis exhibiting a range of erythropoietin levels were screened for the JAK2 V617F mutation. One patient (1.6%) was found to have this mutation, and has remained stable for 9 years, suggesting that the JAK2 V617F mutation is rare in patients with idiopathic erythrocytosis.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis.

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the alpha-subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel-Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Red cell enzymes.

Mutations leading to red cell enzyme deficiencies can be associated with diverse phenotypes that range from hemolytic anemia, methemoglobinemia, polycythemia, and neurological and developmental abnormalities. While most of these mutations occur sporadically, some such as common glucose-6-phosphate dehydrogenase (G6PD) mutants are endemic and rarely cause disease. Common G6PD mutants likely reached their prevalence because they provide some protection against severe malarial complications. In this review G6PD, pyruvate kinase, 5' nucleotidase, and cytochrome b5 reductase deficiencies will be discussed in greater detail. Limitations of commonly used screening tests for detection of these disorders will also be emphasized, as well as emerging knowledge about non-enzymatic function of the glycolytic enzymes.

Erythrocytosis due to bisphosphoglycerate mutase deficiency with concurrent glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.

A 28-year-old asymptomatic male of Iranian Jewish (Meshadi) heritage was found on routine exam to have an erythrocytosis (RBC = 6.22 x 10(12)/l, Hgb = 19.2 g/dl, Hct = 58.9%). Splenomegaly was absent on physical exam. There was no family history of erythrocytosis. His oxygen dissociation curve was left-shifted with a p50 of 19 mmHg (normal = 25-32 mmHg). Hemoglobin electrophoresis showed no abnormalities. DNA sequencing of the hemoglobin beta globin gene and both alpha globin genes did not reveal a mutation. A 2,3-bisphosphoglycerate (BPG) level was markedly decreased at 0.3 micromol/g Hb (normal = 11.4-19.4 micromol/g Hb). The patient's bisphosphoglycerate mutase (BPGM) enzyme activity was also markedly decreased at 0.16 IU/g Hb (normal = 4.13-5.43 IU/g Hb). A red cell enzyme panel revealed a markedly decreased G-6-PD level (0.3 U/g Hb, normal = 8.6-18.6 U/g Hb). His parents and a brother were also available for evaluation. Both parents showed normal 2,3-BPG levels but BPGM activity approximately 50% of normal. Paradoxically, the brother showed normal BPGM activity but a slightly decreased 2,3-BPG level. All family members had markedly decreased G-6-PD activity. DNA sequencing of the BPGM gene showed the propositus to be homozygous for 185 G-->A, Arg 62 Gln in exon 2. Thus, the erythrocytosis in this patient is secondary to low 2,3-BPG levels, due to a deficiency in BPG mutase. This appears due to consanguinity within this family.

Effects of chronic nitric oxide synthase inhibition on renal function and histology in polycythemic rats.

Augmented endogenous nitric oxide (NO) production may ameliorate derangement of renal functions or glomerular damage in polycythemia. To investigate this possibility, we examined the effect of NO synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME; 50 mg/dl in drinking water) on renal functions and histology in heminephrectomized Sprague-Dawley rats treated for 4 weeks with recombinant human erythropoietin (rh-EP; 500 IU/kg on alternate days). L-NAME elevated the blood pressure which was aggravated by concomitant rh-EP and was ameliorated by treatment with a nonpeptide angiotensin type 1 receptor blocker (CV116; 60 mg/kg in chow). The hematocrit level was prominently increased by rh-EP. The glomerular filtration rate was impaired by L-NAME alone, but was maintained by concomitant administration of rh-EP or CV116. Micropuncture experiments revealed that the glomerular capillary pressure was similarly elevated by L-NAME alone or in combination with rh-EP. L-NAME significantly, although not prominently, aggravated glomerular sclerosis observed with rh-EP alone, and concomitant CV116 ameliorated the glomerular damage. These results suggest that, in polycythemia, enhanced NO production buffers the glomerular damage, and the balance between NO and angiotensin II may play an important role in maintaining renal function and glomerular structure.

Red cell lipid peroxidation and antioxidant enzymes in iron deficiency.

Whether iron deficient RBC in humans have a reduced, or an increased, susceptibility to lipid peroxidation was studied in the iron deficiency states of primary proliferative polycythaemia and iron deficiency anaemia and related to changes in the activities of iron-dependent and non-iron dependent antioxidant enzymes. Susceptibility of RBCs to lipid peroxidation was increased when expressed per g Hb. However, this was a result of the low RBC Hb giving an increased membrane lipid: Hb ratio in the incubations. Results were normal when expressed either per cell, or per ml, RBC. Glutathione reductase was normal. Increased RBC superoxide dismutase activity in iron deficiency may be explained by the younger RBC population and reductions in glutathione peroxidase and catalase activities by the microcytic hypochromic changes and the lack of availability of iron, respectively. There is no evidence of an increased susceptibility of RBC to lipid peroxidation in iron deficiency.

Urinary N-acetyl-beta-D-glucosaminidase activity in healthy, polycythemic and hypoxic neonates.

The authors investigated the urinary N-acetyl-beta-D-glucosaminidase (NAG) activity in the case of 101 normal healthy and 20 polycythemic newborns and prematures, and 50 prematures suffering from hypoxia on the 1st, 2nd, 4th, 14th, and 28th day after birth. The obtained activities were referred to the creatinine concentrations of the urine samples and given as NAG index. There were no significant differences in the NAG indices either between fullterm and preterm babies or between appropriate for gestational age (AGA) and small for gestational age (SGA) neonates of the normal group. The NAG indices on the first day of life were significantly higher in the case of polycythemic newborn in comparison with the normal group (p less than 0.01). On the 14th day, after the partial plasma exchange, the NAG indices returned to the normal range. The premature babies suffering from IRDS received an average 10.1 days oxygen supplementation. Their NAG indices were significantly (p less than 0.01) higher on the 1st, 2nd, 4th days than those of the healthy prematures of the normal group and decreased considerably up to the 14th day. Finally the NAG indices reached the normal value on the 28th day. These results support the assumption that the urinary NAG index is a suitable indicator of the renal tubular damage during the newborn period.