Transgenic Sickle Cell Mice Research Articles

Differential expression of E- and P-selectin in the microvasculature of sickle cell transgenic mice.

There is a growing body of evidence that endothelial cells assume an inflammatory phenotype in sickle cell disease. The authors determined whether (1) the expression of E- and P-selectin differs between sickle cell transgenic (beta(S)) mice and their wild-type counterparts, and (2) blood platelets and/or neutrophils contribute to the altered selectin expression. Expression of E- and P-selectin was measured in different regional vascular beds of wild-type and beta(S) mice (with or without thrombocytopenia or neutropenia) using the dual radiolabeled monoclonal antibody technique. Constitutive expression of P-selectin was significantly increased in the heart, lungs, small bowel, large bowel, and penis of beta(S) versus WT mice. While thrombocytopenia reduced P-selectin expression in the small bowel and penis of beta(S) mice, neutropenia was associated with a reduction in P-selectin expression only in the penis. E-selectin expression was not significantly elevated in any vascular bed except the penis of beta(S) mice. Sickle cell disease promotes an increased P-selectin expression in several vascular beds. An accumulation of platelets may explain the increased P-selectin expression observed in some vascular beds.

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice.

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.

Differential gene expression in the kidney of sickle cell transgenic mice: upregulated genes

The S+S-Antilles transgenic mouse used in this study has renal defects similar to those seen in sickle cell anemia patients: congested glomeruli, medullary fibrosis, renal enlargement, vasoocclusion, and a urine concentrating defect. We used gene expression microarrays to identify genes highly up-regulated in the kidneys of these mice and validated their expression by real-time PCR. Kidney hypoxia, as demonstrated by the presence of deoxyhemoglobin, was detected by blood oxygen dependent magnetic resonance imaging (BOLD-MRI). Some of the up-regulated genes included cytochrome P450 4a14, glutathione- S-transferase α-1, mitochondrial hydroxymethylglutaryl CoA synthase, cytokine inducible SH-2 containing protein, retinol dehydrogenase type III, arginase II, glycolate oxidase, Na/K ATPase, renin-1, and alkaline phosphatase 2. An increase in enzyme activity was also demonstrated for one of the up-regulated genes (arginase II). These genes can be integrated into several different pathophysiological processes: a hypoxia cascade, a replacement cascade, or an ameliorating cascade, one or all of which may explain the phenotype of this disease. We conclude that microarray technology is a powerful tool to identify genes involved in renal disease in sickle cell anemia and that the identification of various metabolic pathways may open new avenues for therapeutic interventions.

Differential gene expression in the kidney of sickle cell transgenic mice: upregulated genes

The S+S-Antilles transgenic mouse used in this study has renal defects similar to those seen in sickle cell anemia patients: congested glomeruli, medullary fibrosis, renal enlargement, vasoocclusion, and a urine concentrating defect. We used gene expression microarrays to identify genes highly up-regulated in the kidneys of these mice and validated their expression by real-time PCR. Kidney hypoxia, as demonstrated by the presence of deoxyhemoglobin, was detected by blood oxygen dependent magnetic resonance imaging (BOLD-MRI). Some of the up-regulated genes included cytochrome P450 4a14, glutathione- S -transferase α-1, mitochondrial hydroxymethylglutaryl CoA synthase, cytokine inducible SH-2 containing protein, retinol dehydrogenase type III, arginase II, glycolate oxidase, Na/K ATPase, renin-1, and alkaline phosphatase 2. An increase in enzyme activity was also demonstrated for one of the up-regulated genes (arginase II). These genes can be integrated into several different pathophysiological processes: a hypoxia cascade, a replacement cascade, or an ameliorating cascade, one or all of which may explain the phenotype of this disease. We conclude that microarray technology is a powerful tool to identify genes involved in renal disease in sickle cell anemia and that the identification of various metabolic pathways may open new avenues for therapeutic interventions.

Niprisan (Nix-0699) improves the survival rates of transgenic sickle cell mice under acute severe hypoxic conditions.

The substitution of glutamic acid by valine at the sixth position of the beta-globins of haemoglobin S (Hb S) causes a drastic reduction in the solubility of the deoxy form of Hb S. Under hypoxic conditions, deoxy-Hb S molecules polymerize inside the cells, forming rigid, sickled cells. We studied the effect of Niprisan (Nix-0699), a naturally occurring antisickling agent, on the survival of transgenic (Tg) sickle mice under severe acute hypoxic conditions (60 min). Before hypoxia exposure, the mice were treated by gavage once daily for 7 d with 0 mg/kg (n = 10), 10 mg/kg (n = 5), 50 mg/kg (n = 5), 300 mg/kg (n = 4) or 500 mg/kg (n = 5) of Nix-0699. The mean survival times of the untreated and treated mice were 10, 25, 39, 55 or 60 min respectively. The percentage of sickled cells in the venous blood of the treated mice was lower than that in control mice and was dose dependent. Histological examination of the lungs of the control mice showed entrapment of massive numbers of sickled cells in the alveolar capillaries, although the degree of such entrapment decreased with the increased dose of Nix-0699. Nix-0699 may be a promising option for the treatment and management of patients with sickle cell disease.

Inhibition of nitric oxide synthase ameliorates cellular injury in sickle cell mouse kidneys

In previous studies of transgenic sickle cell mice, increased renal expression of inducible nitric oxide synthase (iNOS) and endothelial cell isoform of NOS (EcNOS) was found by Western blot and immunohistochemistry. In addition, putative evidence of peroxynitrite (ONOO-) formation was found in the form of positive immunostaining and immunoblot for nitrotyrosine. Apoptosis was also detected by DNA strand breakage and TUNEL assay. The present study was carried out to examine the role of NO/ONOO- in mediating renal tubular cell apoptosis in sickle cell mouse kidneys. Mercaptoethylguanidine (MEG), a compound that selectively inhibits iNOS and also is a scavenger of ONOO-, was administered intraperitoneally over a five-day period to control and betas mice. Immunohistochemistry of iNOS and nitrotyrosine, DNA electrophoresis, ApoTACS assay for apoptosis, and Western blot of poly(ADP-ribose) polymerase (PARP) were carried out. MEG administration virtually eliminated renal immunostaining of iNOS and nitrotyrosine and prevented DNA strand breakage. In addition, Western blot analysis of PARP, a nuclear DNA-reparative enzyme activated in response to DNA strand breakage, was found to be cleavaged in hypoxic betas mice, but was partially protected in MEG-treated betas hypoxic mice. Finally, apoptosis was markedly reduced by MEG in betas hypoxic mice. These observations provide evidence that NO and/or ONOO- are responsible for initiating cell damage, which leads to apoptosis in sickle cell mouse kidneys.

In vivo blood flow abnormalities in the transgenic knockout sickle cell mouse.

The accepted importance of circulatory impairment to sickle cell anemia remains to be verified by in vivo experimentation. Intravital microscopy studies of blood flow in patients are limited to circulations that can be viewed noninvasively and are restricted from deliberate perturbations of the circulation. Further knowledge of sickle blood flow abnormalities has awaited an animal model of human sickle cell disease. We compared blood flow in the mucosal-intestinal microvessels of normal mice with that in transgenic knockout sickle cell mice that have erythrocytes containing only human hemoglobin S and that exhibit a degree of hemolytic anemia and pathological complications similar to the human disease. In sickle cell mice, in addition to seeing blood flow abnormalities such as sludging in all microvessels, we detected decreased blood flow velocity in venules of all diameters. Flow responses to hyperoxia in both normal and sickle cell mice were dramatic, but opposite: Hyperoxia promptly slowed or halted flow in normal mice but markedly enhanced flow in sickle cell mice. Intravital microscopic studies of this murine model provide important insights into sickle cell blood flow abnormalities and suggest that this model can be used to evaluate the causes of abnormal flow and new approaches to therapy of sickle cell disease.

Peroxynitrite formation and apoptosis in transgenic sickle cell mouse kidneys

In a previous study, nitric oxide synthases (NOS) were found to be strongly expressed in the tubular epithelium of kidneys of a transgenic mouse model of sickle cell disease (alphaHbetaS[betaMDD]). Because NOS activity is often associated with peroxynitrite formation when superoxide radical (.O-2) is present in abundance, we examined the kidneys of sickle cell mice for nitrotyrosine, considered to be a footprint of ONOO-. Western blot and immunohistochemistry for nitrotyrosine was carried out. Since peroxynitrite and other reactive oxygen radicals are capable of causing apoptosis, we also performed agarose gel electrophoresis of kidney DNA and TUNEL staining of nuclei, indicators of apoptosis. Nitration of tyrosine residues of three proteins (kD 66, 57 and 22) was found on Western blot of kidney protein extracts of the sickle cell mice. The degree of tyrosine nitration of the 66 kD protein was not significantly different in the control versus transgenic mice, whereas tyrosine nitration of the 57 and 22 kD proteins was clearly increased in transgenic mice. Strong immunostaining for nitrotyrosine was seen in tubular epithelial cells of the sickle cell mice, in close proximity to positive immunostaining of iNOS. Neither iNOS nor nitrotyrosine was expressed in the control mice. DNA "laddering" was found localized to the same zones of the kidney as nitrotyrosine and iNOS immunostaining. TUNEL assay on mouse kidney tissue sections showed minimal tubular cell apoptosis in normal mouse with hypoxia, mild tubular cell apoptosis in sickle cell mouse in room air, and moderate tubular cell apoptosis in sickle cell mouse with hypoxia. The observations suggest that ONOO- and perhaps other reactive oxygen species are being produced in the sickle cell kidney. The mechanism may be ischemia/reperfusion due to intermittent vascular occlusion by sickle cells. The resulting hypoxia could result in iNOS activation, superoxide radical and peroxynitrite formation. Two consequences of these reactions appear to be nitration of tyrosine residues of some renal proteins and enhanced apoptosis.

Inhaled Nitric Oxide Prolongs Survival in Transgenic Sickle Cell (SAD) Mice Model During Acute Hypoxia

Inhaled Nitric Oxide Prolongs Survival in Transgenic Sickle Cell (SAD) Mice Model During Acute Hypoxia

Immunohistochemical Localization of Hepatic Nitric Oxide Synthase in Normal and Transgenic Sickle Cell Mice: The Effect of Hypoxia

Nitric oxide (NO) generated from L-arginine and molecular oxygen by nitric oxide synthase (NOS) has been shown to influence hepatocellular function and pathology in response to ischemia and certain hepatotoxins. In the present study, we examined the liver of a transgenic line of sickle cell mice for hepatocellular injury and localization of two isoforms of NOS, the endothelial constitutively expressed isoform (EcNOS) and the inducible isoform (iNOS) by immunohistochemistry. Diffuse expression of EcNOS was observed in hepatocytes of control and sickle cell animals maintained under room air conditions. In contrast, iNOS was observed only in the sickle cell mice, well-localized to hepatocytes surrounding the central veins of the lobules. When normal mice were exposed to hypoxic conditions for 4 to 5 days, iNOS immunostaining appeared de novo in a patchy distribution throughout the liver lobules. In the sickle cell mice, hypoxia appeared to increase the subjective intensity of pericentral staining of iNOS. Liver histology was normal in the sickle cell mice maintained under room air conditions, but showed multifocal areas of necrosis when sickling was exacerbated by chronic hypoxic conditions. However, a pericentral zone of preserved architecture was present, corresponding to the region of iNOS staining. We postulate that pericentral induction of iNOS under ambient conditions occurs in transgenic sickle cell mice in response to particularly intense hypoxic conditions near the central veins of the liver. Increases in NO synthesis may occur in this region, which would serve to protect these cells from ischemic damage either directly or by maintaining blood flow. These findings could be relevant to liver pathophysiology in patients with sickle cell disease.

Renal nitric oxide synthases in transgenic sickle cell mice

The alpha H beta S [beta MDD] mouse is a useful model for studying renal functional abnormalities in sickle cell disease. We previously reported that these mice develop a urine concentrating defect when chronically exposed to a low oxygen environment. In the present study, we measured glomerular filtration rate (GFR), urinary excretion of NO2 s+ NO3, the stable products of nitric oxide (NO), and the abundance of endothelial constitutive nitric oxide synthase (NOS III) and inducible nitric oxide synthase (NOS II) in the kidneys by Western blot. Immunohistochemistry was also carried out. We found that GFR is significantly higher in the transgenic mice than in controls. The urinary NO2 + NO3/creatinine ratio was also higher. The Western blots revealed that both NOS III and NOS II are markedly increased in the kidneys of transgenic mice as compared to normal control mice. Immunohistochemistry localized NOS III reactivity in proximal convoluted cells in the cortex of control and alpha H beta S [beta MDD] mice. NOS II immunostaining was not seen in control mice but was clearly evident in glomeruli and distal nephron segments of the alpha H beta S [beta MDD] mice. These observations suggest that NOS II is induced in glomeruli and distal nephrons of the alpha H beta S [beta MDD] mice. An increase in synthesis of NO may occur in the glomeruli as a result of NOS II induction, and this may contribute to the hyperfiltration in these mice.