ALBERT

Description: The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 μg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties.

Description: Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Description: Abstract 1637 The mechanisms underlying sickle red blood cell (RBC) adhesion to the endothelium, which constitutes a major pathologic event in sickle cell disease (SCD), are not fully understood. Adhesion of sickle RBCs to endothelial cells is believed to be regulated by multiple hematologic and physiologic factors including fetal hemoglobin levels, leukocyte numbers, oxygen tension, inflammatory cytokines, and nitric oxide (NO) bioavailability, but the extent to which each parameter contributes to sickle RBC adhesion remains unclear. Here we studied the effects of hypoxia and NO bioavailability on sickle RBC adhesion using mice deficient for endothelial nitric oxide synthase (eNOS). We found that these mice had NO metabolite levels comparable to those of SCD UAB mice (Ryan et al. Science 278:873, 1997). Sickle RBCs obtained from SCD UAB mice or control RBCs from control mice (C57BL/6J) were injected into eNOS-deficient or control mice. Sickle RBC adhesion in the skull bone marrow venules was studied by intravital microscopy. Control RBCs did not show any adherence to the endothelium in either control mice or eNOS-/- mice and only sickle RBCs adhered to the endothelium in eNOS-/- mice. Sickle RBCs adhered to endothelial cells in control mice under normoxia at an adhesion score of 0.6, which was enhanced to 2.7 by hypoxia, suggesting that hypoxia increases sickle RBC adhesion. In contrast, in eNOS-/- mice, the adhesion score of sickle RBCs was 2.5 under normoxia and increased to 13.7 under hypoxia, indicating a synergistic enhancement of sickle RBC adhesion under hypoxia and low NO availability. To examine whether increasing NO bioavailability in eNOS-deficient mice is sufficient for inhibiting sickle RBC adhesion, we treated eNOS-/- mice under hypoxia with low-dose NO (20 ppm). Inhalation of low-dose NO (20 ppm) restored reduced NO metabolite levels in eNOS-/- mice and decreased sickle RBC adhesion scores under hypoxia to levels which were statistically indistinguishable from those of eNOS-/- mice under normoxia and comparable to those of control mice under hypoxia. These results suggest that NO inhalation disrupted sickle RBC adhesion by hypoxia and low NO bioavailability. Moreover, we showed that inhaled NO induced only a marginal change in wall shear rates which exert effects on blood cell-endothelial cell interactions, indicating that inhaled NO inhibits sickle RBC adhesion under hypoxic conditions by both wall shear rate-dependent and -independent mechanisms. These in vivo studies demonstrate that sickle RBC adhesion is regulated in a synergistic manner by hypoxia and NO bioavailability, and that inhaled NO inhibits sickle RBC adhesion in part through modulation of wall shear rates. Inhalation of NO may alleviate the clinical severity of SCD patients by interrupting the synergy created by hypoxia and reduced NO availability for sickle RBC adhesion. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Adhesion of sRBCs is synergistically regulated by hypoxia and low NO bioavailability. P-selectin and p38 kinase pathways play a role in the synergistic adhesion of sRBCs.

Description: Painful vaso-occlusive episodes are a hallmark of sickle cell disease (SCD), an inherited blood disorder caused by a point mutation in the β-globin gene. Vaso-occlusive events account for the majority of SCD-related hospital admissions and contribute importantly to major organ damage and decreased survival. Therapies to prevent and treat acute vaso-occlusive episodes are limited and there is a need for new treatment options. Adhesive interactions between circulating sickle red blood cells (sRBC), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusive episodes. These cell adhesive interactions further manifest with increased local and systemic inflammation and activated coagulation evidenced in patients and mouse models of SCD. Anti-thrombin III (ATIII) is an endogenous inhibitor of thrombin and other serine proteases in the coagulation pathway. Apart from the role in hemostasis, ATIII exerts strong anti-inflammatory properties. Plasma levels of ATIII and ATIII activity are decreased in SCD and further decline during vaso-occlusive crisis. These findings indicate that ATIII deficiency may contribute by multiple mechanisms to the pathophysiology of acute vaso-occlusion and that repletion therapy with ATIII may provide a novel, multi-targeted therapeutic approach to this difficult-to-treat complication. In this study, we examined whether ATIII supplementation is able to reduce adhesion of sRBC and leukocytes to vascular endothelial cells in the calvarial bone marrow microvasculature in a mouse knockout-transgenic mouse model of SCD (Ryan, 1997). Homozygous SCD mice were injected intravenously with 230 U/kg ATIII (Thrombate III; Grifols, USA) or vehicle (saline). After 48 hours, the mice were subjected to 3 hours of hypoxia (8% O2 in medical air) followed by 3 hours of reoxygenation at room air (21% O2). Intravital microscopy analysis of the bone marrow microcirculation was performed to monitor adhesive interactions between blood cells and vascular endothelium. Leukocytes were labeled in vivo with phycoerythrin-conjugated anti-CD45 antibody which was infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery to the recipient mice. Effects of ATIII on sRBC and leukocyte adhesive interactions were compared to vehicle. Administration of ATIII to SCD mice resulted in more than 5 fold decrease in sRBC adhesion (p

Description: Abstract 1622 Despite considerable concerns and efforts, the mechanism of action of hydroxyurea (HU) for the induction of fetal hemoglobin (HbF) remains elusive. For example, clinical studies with HU suggest that bone marrow reserve is critical for HbF response to HU, but the underlying mechanism remains unknown. We and others have demonstrated that HU activates the cGMP signaling pathway in erythroid cells, which plays a role in HbF induction. However, the mechanisms by which intracellular signals are transduced to downstream cascades of cyclic nucleotide-dependent pathways in erythroid cells treated with HU remain to be established. Here we present evidence that HU induces HbF expression by activating the cAMP signaling pathway through two independent mechanisms: cAMP and cGMP. To study signal transduction by HU in cyclic nucleotide-dependent pathways, we initially focused on identifying substrates for cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) that are expressed in erythroid-lineage cells. We found that vasodilator-stimulated phosphoprotein (VASP), which is a 46/50-kDa phosphoprotein expressed in platelets at high levels, is also expressed in erythroid-lineage cells. VASP can be phosphorylated by cyclic nucleotide-elevating agents such as forskolin (activator of adenylate cyclase) and nitric oxide donors (activator of soluble guanylate cyclase). Interestingly, cAMP and cGMP phosphorylate distinct serine residues of VASP; Ser157 is phosphorylated by cAMP-elevating agents, while cGMP-elevating agents phosphorylated Ser239. Although HU increased both intracellular cAMP and cGMP levels in CD34+-derived erythroblasts, we found that Ser157, but not Ser239, is phosphorylated in adult erythroid cells treated with HU, suggesting activation of the cAMP signaling pathway. However, HU-induced HbF expression was down-regulated by inhibiting the activity of adenylate cyclase or soluble guanylate cyclase, suggesting that both enzymes are involved in HU-induced HbF expression. Our studies found that HU decreased the expression of cGMP-inhibitable phosphodiesterase 3B in a manner dependent on soluble guanylate cyclase, resulting in activation of the cAMP signaling pathway. Although a recent study showed that HU directly activates soluble guanylate cyclase, our studies showed that HU is unable to directly stimulate the enzyme activity of adenylate cyclase. Furthermore, HU induced the expression of cyclooxygenase-1 (COX-1) and increased the production of prostaglandin E2 (PGE2) that activates adenylate cyclase through G protein-coupled E-prostanoid receptors. Plasma PGE2 levels were also elevated in sickle cell patients upon HU therapy. These results demonstrate that HU induces HbF expression by activating the cAMP pathway by cAMP- and cGMP-dependent mechanisms, producing redundancy in the response of HbF to HU. Both cAMP and cGMP may represent major molecules that transduce signals from HU to the fetal globin gene. It is known that non-erythroid cells such as leukocytes and monocytes produce a large amount of PGE2. Thus, the involvement of PGE2 in HU-induced HbF expression may suggest an important role of non-erythroid cells as well as bone marrow reserve in the induction of HbF expression. More interestingly, several single nucleotide polymorphisms with amino acid changes have been demonstrated for COX-1; some genetic variants exhibit reduced COX activities. If SCD patients have some mutations in the COX-1 gene, such patients might be resistant to HU therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Objective:Recurrent acute episodes of vaso-occlusion (VOC) are one of the hallmarks of sickle cell disease (SCD). Leukocytes are in an activated state in SCD and their recruitment to the vascular endothelium marks a critical event in the pathophysiology of the vaso-occlusive process. Endothelin-1 (ET-1), a potent vasoconstrictive and pro-inflammatory endogenous peptide, is elevated in steady state SCD, increases during acute VOC crisis, and remains elevated post crisis. This study asked whether the ET-1-ETRA signaling pathway is involved in promoting leukocyte adhesion in a hypoxia-reoxygenation challenged mouse model of SCD. Secondly, we wondered whether ET-1 is able to directly activate and increase the proadhesive phenotype of the peripheral leukocyte population. Methods: Animals:We used the UAB knockout-transgenic mouse model of SCD for our studies (Ryan, 1997). Experimental groups consisted of homozygous (SCD-/-), heterozygous (SCD+/-), and C57BL/6 control mice. Intravital Microscopy:Bosentan (BOS; dual ETRA/B antagonist) or Ambrisentan (AMB; specific ETRA antagonist) was administered by medicated chow for 8 weeks. Prior to imaging, animals were exposed to 5 hrs of 8% O2 followed by 2 hrs of reoxygenation in room air. Leukocytes were stained in vivo with a PE-conjugated anti-CD45 antibody. Mice were anesthetized and the calvarial bone marrow microvascular network was observed using an intravital microscope with water-immersion objectives. Images were analyzed off-line for leukocyte velocity, adhesion, and flow dynamics (Image Pro-Plus 5.0). Superoxide/Cytokine/Integrin measurements: Leukocytes were isolated from freshly harvested peripheral blood (SCD-/- and controls) and exposed to ET-1 or vehicle. ROS production was measured by luminescence (Lumistar Galaxy Luminometer). Intracellular cytokine and surface integrin expression was analyzed by flow cytometry. Neutrophil Adhesion Assay: Peripheral neutrophils were purified by a Ficoll gradient from SCD-/- mice and controls. Following ex vivo incubation with ET-1 or vehicle, the purified neutrophils were added to pre-activated (thrombin) rat PMVECs. After washing with PBS, the cells were fixed and stained with MPO to identify adherent neutrophils. Results: Intravital microscopy revealed that ETR blockade with BOS, but not AMB, enhanced WBC rolling and adhesion at steady state without altering flow velocity, suggesting an important role for ETRB signaling at baseline in SCD-/- animals. Following a hypoxia/reoxygenation protocol, both antagonists were equally effective in decreasing WBC rolling and adhesion in SCD-/- animals. Both ETR antagonists were able to correct H/R-induced decreases in blood flow velocity to near baseline values. Next we asked if ET-1 is capable of activating WBCs by first measuring levels of receptor mRNA in sorted peripheral neutrophils and monocytes. Next we measured intracellular cytokine, surface integrins and ROS production following ex vivo exposure to ET-1. Results indicated a very low amount of ETRA mRNA in neutrophils of SCD-/- mice and controls. ETRB mRNA was predominantly expressed in monocytes of SCD-/- mice. ET-1 ex vivo stimulation failed to increase cytokine, integrin and ROS levels beyond baseline in sickle neutrophils and monocytes. Functionally, more neutrophils from SCD-/- mice adhered to pre-activated endothelial cells than controls; however, ex vivo ET-1 stimulation failed to increase adhesion. Summary and Conclusions: Our results demonstrate that selective ETRA blockade is sufficient to abrogate H/R-induced leukocyte-endothelial interactions in a preclinical model of SCD. While the dual ETRA/B antagonist produced similar results after H/R, its proadhesive effect at steady state suggests an important role for the endothelin B receptor under baseline conditions. Although the long-term significance of these findings remains to be elucidated, at this early stage our results suggest a potential therapeutic advantage for a selective ETRA treatment strategy in SCD. The lack of response of the leukocyte population to direct ET-1 stimulation suggests that ET-1 targets primarily the vascular endothelium to promote leukocyte adhesion. In conclusion, this study established a potentially important role for clinically available ETRA antagonists in the treatment portfolio for patients with SCD. Disclosures Gutsaeva: Grifols: Research Funding.

Description: Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3’-UTR (3’-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.

Description: Abstract 1545 Poster Board I-568 To date, a variety of therapeutic approaches to sickle cell disease (SCD) have been explored, however, knowledge about the efficacy of anti-adhesive agents in this disorder is limited. In SCD, P-selectin expressed on endothelial cells plays a key role in leukocyte recruitment as well as in the adhesion of sickle red blood cells (sRBC) to the endothelium. The interaction of P-selectin and its ligands is thus likely to contribute to impairment of the microvascular flow and thereby to the development of painful vaso-occlusive episodes. Studies with anti-P-selectin antibody and P-selectin-deficient mice support the notion that P-selectin-directed interventions may be a potential approach to the treatment of vaso-occlusive episodes in SCD. Aptamers, short single-stranded oligonucleotides, have been developed for a wide range of therapeutic targets (Keefe & Schaub, 2008). Aptamers bind molecular targets with high affinity and specificity, do not elicit immune responses, and can be readily delivered to the vascular compartment intravenously or subcutaneously. Although anti-P-selectin aptamers have been shown to inhibit leukocyte rolling in vitro and to display efficacy in mouse models for inflammation, the anti-adhesion activity of anti-P-selectin aptamers has never been evaluated in SCD. The purpose of this study was to determine in vivo whether the anti-P-selectin aptamer ARC5690 can inhibit adhesion of sickle RBC and leukocytes to vascular endothelial cells in the bone marrow microvasculature of SCD model mice. Knockout-transgenic SCD mice generated by Ryan et al. (Ryan et al., 1997) were used in this study. SCD mice were injected with 20 mg/kg ARC5690, scrambled aptamer ARC5694 (negative control), or vehicle (saline). Anti-P-selectin monoclonal antibody was also used as a positive control. After 2.5 hours, the mice were subjected to 1 hour of hypoxia (12% O2 in air) followed by 1 hour of reoxygenation at room air. Intravital observations of the bone marrow microcirculation were performed to monitor adhesive interactions between blood cells and endothelial cells. Leukocytes were labeled in vivo with rat anti mouse CD45 antibody conjugated with PE which were infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery. Effects of ARC5690 on sRBC and leukocyte adhesive interactions were compared to those of ARC5694 and vehicle. Administration of the anti-P-selectin aptamer ARC5690 significantly reduced sRBC adhesion (p