Effect of fetal hemoglobin on microvascular regulation in sickle transgenic-knockout mice.

In sickle cell disease, intravascular sickling and attendant flow abnormalities underlie the chronic inflammation and vascular endothelial abnormalities. However, the relationship between sickling and vascular tone is not well understood. We hypothesized that sickling-induced vaso-occlusive events and attendant oxidative stress will affect microvascular regulatory mechanisms. In the present studies, we have examined whether microvascular abnormalities expressed in sickle transgenic-knockout Berkeley (BERK) mice (which express exclusively human alpha- and beta(S)-globins with <1% gamma-globin levels) are amenable to correction with increased levels of antisickling fetal hemoglobin (HbF). In BERK mice, sickling, increased oxidative stress, and hemolytic anemia are accompanied by vasodilation, compensatory increases in eNOS and COX-2, and attenuated vascular responses to NO-mediated vasoactive stimuli and norepinephrine. The hypotension and vasodilation (required for adequate oxygen delivery in the face of chronic anemia) are mediated by non-NO vasodilators (i.e., prostacyclin) as evidenced by induction of COX-2. In BERK mice, the resistance to NO-mediated vasodilators is associated with increased oxidative stress and hemolytic rate, and in BERK + gamma mice (expressing 20% HbF), an improved response to these stimuli is associated with reduced oxidative stress and hemolytic rate. Furthermore, BERK + gamma mice show normalization of vessel diameters, and eNOS and COX-2 expression. These results demonstrate a strong relationship between sickling and microvascular function in sickle cell disease.

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion.

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants.

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).

Robust vascular protective effect of hydroxamic acid derivatives in a sickle mouse model of inflammation.

OBJECTIVE: Clinically, the vascular pathobiology of human sickle cell disease includes an abnormal state of chronic inflammation and activation of the coagulation system. Since these biologies likely underlie development of vascular disease in sickle subjects, they offer attractive targets for novel therapeutics. Similar findings characterize the sickle transgenic mouse, which therefore provides a clinically relevant inflammation model. METHOD: The authors tested two polyhydroxyphenyl hydroxamic acid derivatives, didox and trimidox, in sickle transgenic mice. Animals were examined by intravital microscopy (cremaster muscle and dorsal skin fold preparations) and by histochemistry before and after transient exposure to hypoxia, with versus without preadministration of study drug. Previous studies have validated the application of hypoxia/reoxygenation to sickle transgenic mice as a disease-relevant model. RESULTS: Animals pretreated with these agents exhibited marked improvements in leukocyte/ endothelial interaction, hemodynamics and vascular stasis, and endothelial tissue factor expression. Thus, these drugs unexpectedly exert powerful inhibition on both the inflammation and coagulation systems. CONCLUSIONS: Each of these changes is expected to be therapeutically beneficial in systemic inflammatory disease in general, and in sickle disease in particular. Thus, these novel compounds offer the advantage of having multiple therapeutic benefits in a single agent.

Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation.

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.

Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice.

In sickle cell disease, inflammatory activation of vascular endothelium and increased leukocyte-endothelium interaction may play an important role in the occurrence of vasoocclusion. In sickle mouse models, inflammatory stimuli (e.g., hypoxia-reoxygenation and cytokines) result in increased leukocyte recruitment and can initiate vasoocclusion, suggesting that anti-inflammatory therapy could be beneficial in management of this disease. We have tested the hypothesis that inhibition of endothelial activation in a transgenic mouse model by anti-inflammatory agents would lead to reduced leukocyte recruitment and improved microvascular blood flow in vivo. In transgenic sickle mice, hypoxia-reoxygenation resulted in greater endothelial oxidant production than in control mice. This exaggerated inflammatory response in transgenic mice, characterized by increased leukocyte recruitment and microvascular flow abnormalities, was significantly attenuated by antioxidants (allopurinol, SOD, and catalase). In contrast, control mice exhibited a muted response to antioxidant treatment. In addition, hypoxia-reoxygenation induced activation of NF-kappaB in transgenic sickle mice but not in control mice. In transgenic sickle mice, sulfasalazine, an inhibitor of NF-kappaB activation and endothelial activation, attenuated endothelial oxidant generation, as well as NF-kappaB activation, accompanied by a marked decrease in leukocyte adhesion and improved microvascular blood flow. Thus targeting oxidant generation and/or NF-kappaB activation may constitute promising therapeutic approaches in sickle cell disease.