Therapeutic inhibition of HIF-2α reverses polycythemia and pulmonary hypertension in murine models of human diseases.

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous VhlR200W mutation. Moreover, the aged VhlR200W mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in VhlR200W, Irp1-KO, and double-mutant VhlR200W;Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in VhlR200W, Irp1-KO, and VhlR200W;Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in VhlR200Wmice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

Evaluation of the Effects of Delayed Cord Clamping in Infants of Diabetic Mothers.

OBJECTIVE: This study aimed to investigate the effect of delayed cord clamping (DCC) in infants of diabetic mothers. STUDY DESIGN: Women who had diabetes throughout their pregnancy and gave birth at 37 weeks of gestation or later were included in the study along with their babies. Early cord clamping was performed as soon as possible after birth, while DCC was performed by clamping 60 second after birth. The two groups were compared in terms of venous hematocrit (htc) levels and rates of hypoglycemia, jaundice requiring phototherapy, and respiratory distress. RESULTS: Venous htc levels at postnatal 6 and 24 hours were significantly higher in the DCC group (p = 0.0001). Polycythemia rates were higher in the DCC group at both 6 and 24 hours, but partial exchange transfusion (PET) was not needed in either group. There were no differences between the groups with regard to the rates of hypoglycemia or jaundice requiring phototherapy. Rate of admission to the neonatal intensive care unit (NICU) was lower in the DCC group. CONCLUSION: Although DCC increased the rate of polycythemia, it did not result in PET requirement. Moreover, DCC reduced the severity of respiratory distress and the rate of admission to NICU due to respiratory distress.

Twin-twin transfusion and laser therapy.

PURPOSE OF REVIEW: Twin-to-twin transfusion syndrome (TTTS) is an uncommon, but dangerous, complication of monochorionic diamniotic twin gestations. The purpose of this review is to provide an update on the evolving treatments in TTTS as it pertains primarily to laser photocoagulation, as well as to provide recently published information on outcomes. RECENT FINDINGS: The Solomon laser technique, in which selective fetoscopic laser photocoagulation is first performed and then followed by laser of the vascular equator from one side of the placenta to the other, reduces TTTS complications of twin anemia-polycythemia syndrome and recurrent TTTS. The addition of fetal echocardiography to the historical staging of TTTS adds important information that may guide future therapies. The postlaser ablation rate of neurodevelopmental delay in TTTS has recently been reported to be 14%. Cotwin demise is a significant complication of untreated TTTS and survival carries a 25% risk of cystic periventricular leukomalacia, middle cerebral artery infarction, and injury to other central nervous system structures as noted by neuroimaging. SUMMARY: Laser therapy for TTTS is clearly the only therapy that halts the disease process, allows both fetuses an opportunity to survive and protects a surviving cotwin in the event of the demise of one twin. Laser techniques have evolved greatly over the last 25 years and recent reports with the addition of the Solomon technique appearing to reduce some postlaser complications (twin anemia-polycythemia sequence and recurrent TTTS). Future focus of TTTS therapy should be centered on understanding the pathophysiology of the disease better with improvement in staging of the disease and on comparison of different laser techniques with the overall goal of not only increasing twin survival rates but also reducing long term neurodevelopmental morbidity.

Effect of early versus delayed cord clamping on hematological status of preterm infants at 6 wk of age.

OBJECTIVE: To compare the effect of early cord clamping (ECC) vs. delayed cord clamping (DCC) on hematocrit and serum ferritin at 6 wk of life in preterm infants. METHODS: This randomized controlled trial was conducted in the delivery room and neonatal intensive care unit of a tertiary hospital. One hundred preterm infants born between 30 (0)/7 and 36 (6)/7 wk were randomized to either early or delayed cord clamping groups. Parental informed consent was obtained prior to the delivery. In the ECC group, the cord was clamped immediately after the delivery of the baby and in the DCC group; the cord was clamped beyond 2 min after the baby was delivered. Hematocrit and serum ferritin at 6 wk of life were the primary outcomes. Incidence of anemia, polycythemia and significant jaundice were the main secondary outcomes. RESULTS: The mean hematocrit (27.3 ± 3.8 % vs. 31.8 ± 3.5 %, p value 0.00) and the mean serum ferritin (136.9 ± 83.8 ng/mL vs. 178.9 ± 92.8 ng/mL, p value 0.037) at 6 wk of age were significantly higher in the infants randomized to DCC group. The hematocrit on day 1 was also significantly higher in the DCC group (50.8 ± 5.2 % vs. 58.5 ± 5.1 %, p value 0.00). The DCC group required significantly longer duration of phototherapy (55.3 ± 40.0 h vs. 36.7 ± 32.6 h, p value 0.016) and had a trend towards higher risk of polycythemia. CONCLUSIONS: Delaying the cord clamping by 2 min, significantly improves the hematocrit value at birth and this beneficial effect continues till at least 2nd mo of life.

Fetoscopic laser ablation of placental anastomoses in twin-twin transfusion syndrome using 'Solomon technique'.

OBJECTIVE: To document perinatal outcomes following use of the 'Solomon technique' in the selective photocoagulation of placental anastomoses for severe twin-twin transfusion syndrome (TTTS). METHODS: Between January 2010 and July 2012, data were collected from 102 consecutive monochorionic twin pregnancies complicated by severe TTTS that underwent fetoscopic laser ablation at four different centers. We compared outcomes between subjects that underwent selective laser coagulation using the Solomon technique (cases) and those that underwent selective laser coagulation without this procedure (controls). RESULTS: Of the 102 pregnancies examined, 26 (25.5%) underwent the Solomon technique and 76 (74.5%) did not. Of the 204 fetuses, 139 (68.1%) survived up to 30 days of age. At least one twin survived in 82 (80.4%) pregnancies and both twins survived in 57 (55.9%) pregnancies. When compared with the control group, the Solomon-technique group had a significantly higher survival rate for both twins (84.6 vs 46.1%; P < 0.01) and a higher overall neonatal survival rate (45/52 (86.5%) vs 94/152 (61.8%); P < 0.01). Use of the Solomon technique remained independently associated with dual twin survival (adjusted odds ratio (aOR), 11.35 (95% CI, 3.11-53.14); P = 0.0007) and overall neonatal survival rate (aOR, 4.65 (95% CI, 1.59-13.62); P = 0.005) on multivariable analysis. There were no cases of recurrent TTTS or twin anemia-polycythemia sequence (TAPS) in the Solomon-technique group. CONCLUSIONS: Use of the Solomon technique following selective laser coagulation of placental anastomoses appears to improve twin survival and may reduce the risk of recurrent TTTS and TAPS. Our data support the idea of performing a randomized controlled trial to evaluate the effectiveness of the Solomon technique.

Protective effect of total flavonoids of seabuckthorn (Hippophae rhamnoides) in simulated high-altitude polycythemia in rats.

Seabuckthorn (Hippophae rhamnoides L.) has been used to treat high altitude diseases. The effects of five-week treatment with total flavonoids of seabuckthorn (35, 70, 140 mg/kg, ig) on cobalt chloride (5.5 mg/kg, ip)- and hypobaric chamber (simulating 5,000 m)-induced high-altitude polycythemia in rats were measured. Total flavonoids decreased red blood cell number, hemoglobin, hematocrit, mean corpuscular hemoglobin levels, span of red blood cell electrophoretic mobility, aggregation index of red blood cell, plasma viscosity, whole blood viscosity, and increased deformation index of red blood cell, erythropoietin level in serum. Total flavonoids increased pH, pO2, Sp(O2), pCO2 levels in arterial blood, and increased Na⁺, HCO3⁻, Cl⁻, but decreased K⁺ concentrations. Total flavonoids increased mean arterial pressure, left ventricular systolic pressure, end-diastolic pressure, maximal rate of rise and decrease, decreased heart rate and protected right ventricle morphology. Changes in hemodynamic, hematologic parameters, and erythropoietin content suggest that administration of total flavonoids from seabuckthorn may be useful in the prevention of high altitude polycythaemia in rats.

Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor NVP-BSK805.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.

Therapeutic methods used in patients with Eisenmenger syndrome.

Patients with Eisenmenger syndrome form a small percentage of congenital heart disease patients. The rarity of this syndrome, combined with its complex pathophysiology, account for the insufficient understanding of the principles underlying its proper treatment. The main clinical symptoms are: cyanosis due to secondary erythrocytosis, resulting in increased blood viscosity, iron deficiency anemia (enhanced by unnecessary phlebotomies), blood clotting disturbances, heart failure and serious supraventricular and ventricular arrhythmias. Recent decades have seen developments in pulmonary hypertension pathophysiology which have led to the introduction of new groups of drugs: prostacycline analogs (Epoprostenol, Treprostinil, Beraprost, Illoprost), phosphodiesterase inhibitors (Sildenafil, Tadalafil), endothelin receptor antagonists (Bosentan, Sitaxantan, Ambrisentan) and nitric oxide. These drugs should be administered to patients in III-IV NYHA class. Despite successful early results, the therapeutic effect on patients with Eisenmenger syndrome has not been conclusively established. Our therapeutic efforts should be directed mainly towards preventing complications. As a rule, we should avoid agents with no established therapeutic efficacy and try to alleviate symptoms without any additional risk, so as not to disrupt the existing clinical balance.

An inhibitor of Janus kinase 2 prevents polycythemia in mice.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (<1h) induces mobilization of activated erythroid precursors into the blood, thus allowing drug-response relationships to guide discovery. We conclude that inhibition of Jak2 prevents polycythemia in mice, and furthermore present this model as an efficient tool for the discovery of drugs that effectively treat human PV.

Sustained-release erythropoietin ameliorates cardiac function in infarcted rat-heart without inducing polycythemia.

BACKGROUND: The usefulness of sustained-release erythropoietin for improving left ventricular (LV) function without polycythemia was evaluated in a rat chronic myocardial infarction model. METHODS AND RESULTS: Four weeks after left coronary artery ligation, 50 Sprague-Dawley rats were assigned to 5 groups (n=10, each). Control group had a gelatin sheet (20x20 mm) containing saline applied to the infarct area, whereas the 4 treatment groups had gelatin sheets incorporating erythropoietin 0.1 U, 1 U, 10 U and 100 U, respectively. Endpoint measurements performed at 8 weeks after the coronary ligation revealed that the fractional area change was larger for erythropoietin 1 U and 10 U than in the other 3 groups. The LV end-systolic elastance and the time constant of isovolumic relaxation were better for erythropoietin 1 U and 10 U than in the other 3 groups. The density of vessels larger than 50 microm in diameter was the highest in the erythropoietin 1 U group. The number of red blood cells was significantly increased in groups receiving erythropoietin 10 U and 100 U. CONCLUSIONS: Gelatin hydrogel sheets incorporating 1 U erythropoietin improved LV function without inducing polycythemia in a rat chronic myocardial infarction model.

Angiotensin converting enzyme gene polymorphism and development of post-transplant erythrocytosis.

BACKGROUND: An increased activation of the renin-angiotensin system probably plays a major role in the development of post-transplant erythrocytosis (PTE). It is known that deletion type polymorphism (DD) in the angiotensin converting enzyme (ACE) gene is associated with higher circulating angiotensin II (AII) levels. The aim of this study was to investigate the effect of ACE gene polymorphism on development of PTE. METHODS: 86 PTE patients (male/female: 68/18, mean age: 32 +/- 10 years) and 68 consecutively transplanted non- PTE patients (male/female: 38/30, mean age: 31 +/- 10 years) were included; 140 patients (91%) had been transplanted from living donors; 92 patients (60%) had hypertension. ACE gene polymorphism was determined by polymerase chain reaction (PCR). RESULTS: The mean time to appearance of PTE was 8.8 +/- 7.9 (range of 1-53) months. DD genotype was detected in 65 patients. PTE patients had a higher prevalence of hypertension (70% vs. 46%, p=0.003) and a lower frequency of DD genotype (34% vs. 54%, p=0.014) as compared to non-PTE patients [OR: 2.2 (1.14-4.25, 95% CI)]. PTE developed more frequently in male patients (68/106: 64%) than females (18/48: 38%) (p=0.002). Patients with DD genotype had a significantly longer leading time to PTE in Kaplan-Meier survival analysis with log-rank (136 +/- 15 vs. 92 +/- 13 months, p=0.015). In Cox regression analysis, hypertension (p=0.002) and recipient ACE genotype (p=0.013) were retained as independent variables for predicting PTE development. CONCLUSIONS: PTE develops more frequently in male, hypertensive renal transplant recipients with good allograft function. DD-type ACE gene polymorphism seems to protect against PTE development.

Early cord clamping protects at-risk neonates from polycythemia.

UNLABELLED: Neonatal polycythemia is a potentially lethal, multi-organ disease. We have performed a prospective, open-label study to test the hypothesis that an early cord clamping proximally to the neonate's abdomen could avert from the neonatal circulation a blood volume critical to the occurrence of polycythemia in at-risk neonates. Newborns were divided into group 1 (clamping time within 10 s) and group 2 (clamping time 11-120 s). Group 1 had statistically significant more blood volume sequestered in the cord and less manifestations of polycythemia. CONCLUSION: An early cord clamping is an effective and zero-cost way to prevent polycythemia in at-risk neonates.

[Indications for and modalities of long-term oxygen therapy]. / Indications et modalités de l'oxygénothérapie de longue durée.

Long-term oxygenotherapy treatment prevent pulmonary hypertension and hypercythemia in chronic hypoxic patients. LTOT is indicated in severe patients with a medial survival time of 3.5 years. Medical indication of LTOT is hypoxemia > 55 mmHg, or, if right cardiac sign or polycythemia are present PaO2 between 55 and 60 mmHg. In some cases LTOT is used only during deambulation, if hypoxemia occurs only during activity. In most cases LTOT is received during all the night and as long as possible during the day time to reach 18 hours/24. OLT decreases the risk of polycythemia and pulmonary hypertension and increases the survival rate of chronic hypoxemic patients.

Beta-adrenergic blockade does not prevent polycythemia or decrease in plasma volume in men at 4300 m altitude.

When humans ascend to high altitude (ALT) their plasma volume (PV) and total blood volume (BV) decrease during the first few days. With continued residence over several weeks, the hypoxia-induced stimulation of erythropoietin increases red cell production which tends to restore BV. Because hypoxia also activates the beta-adrenergic system, which stimulates red blood cell production, we investigated the effect of adrenergic beta-receptor inhibition with propranolol on fluid volumes and the polycythemic response in 11 healthy unacclimatized men (21-33 years old exposed to an ALT of 4300 m (barometric pressure 460 Torr) for 3 weeks on Pikes Peak, Colorado. PV was determined by the Evans blue dye method (PVEB), BV by the carbon monoxide method (BVCO), red cell volume (RCV) was calculated from hematocrit (Hct) and BVCO, and serum erythropoietin concentration ([EPO]) and reticulocyte count, were also determined. All determinations were made at sea level and after 9-11 (ALT-10) and 19-20 (ALT-20) days at ALT. At sea level and ALT, six men received propranolol (pro, 240 mg x day[-1]), and five received a placebo (pla). Effective beta-blockade did not modify the mean (SE) maximal values of [EPO] [pla: 24.9 (3.5) vs pro: 24.5 (1.5) mU x ml(-1)] or reticulocyte count [pla: 2.7 (0.7) vs pro: 2.2 (0.5)%]; nor changes in PVEB [pla: -15.8 (3.8) vs pro: -19.9 (2.8)%], RCVCO [pla: +7.0 (6.7) vs pro: + 10.1 (6.1)%], or BVCO [pla: -7.3 (2.3) vs pro: -7.1 (3.9)%]. In the absence of weight loss, a redistribution of body water with no net loss is implied. Hence, activation of the beta-adrenergic system did not appear to affect the hypovolemic or polycythemic responses that occurred during 3 weeks at 4300 m ALT in these subjects.