Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Vasculo-toxic and pro-inflammatory action of unbound haemoglobin, haem and iron in transfusion-dependent patients with haemolytic anaemias.

Increasing evidence suggests that free haem and iron exert vasculo-toxic and pro-inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion-dependent patients with ß-thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic 'free' species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem- and iron-related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble endothelial selectin (sE-selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem-related parameters and soluble intercellular adhesion molecule 1 (sICAM-1), sVCAM-1, interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) with iron-related parameters. While hereditary spherocytosis was associated with the highest IL-6 and TNFα levels, ß-thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions.

Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1ß Release and Cell Death.

Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1ß. Heme-induced IL-1ß release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1ß release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

Inflammation in sickle cell disease.

The primary ß-globin gene mutation that causes sickle cell disease (SCD) has significant pathophysiological consequences that result in hemolytic events and the induction of the inflammatory processes that ultimately lead to vaso-occlusion. In addition to their role in the initiation of the acute painful vaso-occlusive episodes that are characteristic of SCD, inflammatory processes are also key components of many of the complications of the disease including autosplenectomy, acute chest syndrome, pulmonary hypertension, leg ulcers, nephropathy and stroke. We, herein, discuss the events that trigger inflammation in the disease, as well as the mechanisms, inflammatory molecules and cells that propagate these inflammatory processes. Given the central role that inflammation plays in SCD pathophysiology, many of the therapeutic approaches currently under pre-clinical and clinical development for the treatment of SCD endeavor to counter aspects or specific molecules of these inflammatory processes and it is possible that, in the future, we will see anti-inflammatory drugs being used either together with, or in place of, hydroxyurea in those SCD patients for whom hematopoietic stem cell transplants and evolving gene therapies are not a viable option.

Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase-1 induction.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.

Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins.

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4-/- mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function.

There is growing evidence that extracellular haemoglobin and haem mediate inflammatory and oxidative damage in sickle cell disease. Haptoglobin (Hp), the scavenger for free haemoglobin, is depleted in most patients with sickle cell disease due to chronic haemolysis. Although single infusions of Hp can ameliorate vaso-occlusion in mouse models of sickle cell disease, prior studies have not examined the therapeutic benefits of more chronic Hp dosing on sickle cell disease manifestations. In the present study, we explored the effect of Hp treatment over a 3-month period in sickle mice at two dosing regimens: the first at a moderate dose of 200 mg/kg thrice weekly and the second at a higher dose of 400 mg/kg thrice weekly. We found that only the higher dosing regimen resulted in increased haem-oxygenase-1 and heavy chain ferritin (H-ferritin) expression and decreased iron deposition in the kidney. Despite the decreased kidney iron deposition following Hp treatment, there was no significant improvement in kidney function. However, there was a nearly significant trend towards decreased liver infarction.

Not simply misshapen red cells: multimolecular and cellular events in sickle vaso-occlusion.

Thromboinflammatory diseases result from the interactions of vascular endothelial cells, inflammatory cells, and platelets with cellular adhesion molecules, plasma proteins, and lipids. Tipping the balance toward a prothrombotic, proinflammatory phenotype results from multicellular activation signals. In this issue of the JCI, Li et al. explore the regulation of heterotypic neutrophil-platelet contacts in response to TNF-α-induced venular inflammation with relevance to sickle cell disease (SCD).

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

ß-Globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34(+)-based in vitro model.

The ultimate goal of gene therapy for sickle cell anemia (SCA) is an improved phenotype for the patient. In this study, we utilized bone marrow from a sickle cell patient as a model of disease in an in vitro setting for the hyperactive Sleeping Beauty transposon gene therapy system. We demonstrated that mature sickle red blood cells containing hemoglobin-S and sickling in response to metabisulfite can be generated in vitro from SCA bone marrow. These cells showed the characteristic morphology and kinetics of hemoglobin-S polymerization, which we quantified using video microscopy and imaging cytometry. Using video assessment, we showed that delivery of an IHK-ß(T87Q) antisickling globin gene by Sleeping Beauty via nucleofection improves metrics of sickling, decreasing percent sickled from 53.2 ± 2.2% to 43.9 ± 2.0%, increasing the median time to sickling from 8.5 to 9.6 min and decreasing the maximum rate of sickling from 2.3 x 10(-3) sickling cells/total cells/sec in controls to 1.26 x 10(-3) sickling cells/total cells/sec in the IHK-ß(T87Q)-globin group (p < 0.001). Using imaging cytometry, the percentage of elongated sickled cells decreased from 34.8 ± 4.5% to 29.5 ± 3.0% in control versus treated (p < 0.05). These results support the potential use of Sleeping Beauty as a clinical gene therapy vector and provide a useful tool for studying sickle red blood cells in vitro.

Age sensitizes the kidney to heme protein-induced acute kidney injury.

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (~16 mo old), but not young mice (~6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2(+/+) and HO-2(-/-) mice: HO-2(-/-) mice, compared with HO-2(+/+) mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.

Erythroid-specific expression of ß-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-ß-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased ß-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of ß-globin from the integrated SB transgene. IHK-ß-globin mRNA was found in non-erythroid cell types, similar to native ß-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-ß-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-ß-globin transgene for gene therapy of sickle cell disease.

Acadesine inhibits tissue factor induction and thrombus formation by activating the phosphoinositide 3-kinase/Akt signaling pathway.

OBJECTIVE: Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. CONCLUSION: Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Inhaled carbon monoxide reduces leukocytosis in a murine model of sickle cell disease.

Carbon monoxide (CO) has anti-inflammatory properties. We previously reported that acute treatments with inhaled CO inhibit vascular inflammation and hypoxia-induced vasoocclusion in sickle cell disease mouse models. Therefore, we hypothesized that chronic CO inhalation would decrease vascular inflammation and organ pathology in a sickle cell disease mouse model. The treatment of sickle cell disease mice with 25 or 250 parts/million inhaled CO for 1 h/day, 3 days/wk for 8-10 wk significantly decreased the total mean white blood cell, neutrophil, and lymphocyte counts in peripheral blood. Eight weeks of 250 parts/million CO treatments reduced staining for myeloid and lymphoid markers in the bone marrow of sickle mice. Bone marrow from treated sickle mice exhibited a significant decrease in colony-forming unit granulocyte-macrophage during colony-forming cell assays. Anti-inflammatory signaling pathways phospho-Akt and phospho-p38 MAPK were markedly increased in CO-treated sickle livers. Importantly, CO-treated sickle mice had a significant reduction in liver parenchymal necrosis, reflecting the anti-inflammatory benefits of CO. We conclude that inhaled CO may be a beneficial anti-inflammatory therapy for sickle cell disease.

Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy.

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors.

Tumor necrosis factor-alpha-induced accentuation in cryoinjury: mechanisms in vitro and in vivo.

Cryosurgical treatment of solid cancer can be greatly assisted by further translation of our finding that a cytokine adjuvant tumor necrosis factor-alpha (TNF-alpha) can achieve complete cancer destruction out to the intraoperatively imaged iceball edge (-0.5 degrees C) over the current clinical recommendation of reaching temperatures lower than -40 degrees C. The present study investigates the cellular and tissue level dose dependency and molecular mechanisms of TNF-alpha-induced enhancement in cryosurgical cancer destruction. Microvascular endothelial MVEC and human prostate cancer LNCaP Pro 5 (LNCaP) cells were frozen as monolayers in the presence of TNF-alpha. Normal skin and LNCaP tumor grown in a nude mouse model were also frozen at different TNF-alpha doses. Molecular mechanisms were investigated by using specific inhibitors to block nuclear factor-kappaB-mediated inflammatory or caspase-mediated apoptosis pathways. The amount of cryoinjury increased in a dose-dependent manner with TNF-alpha both in vitro and in vivo. MVEC were found to be more cryosensitive than LNCaP cells in both the presence and the absence of TNF-alpha. The augmentation in vivo was significantly greater than that in vitro, with complete cell death up to the iceball edge in tumor tissue at local TNF-alpha doses greater than 200 ng. The inhibition assays showed contrasting results with caspase-mediated apoptosis as the dominant mechanism in MVEC in vitro and nuclear factor-kappaB-mediated inflammatory mechanisms within the microvasculatures the dominant mechanism in vivo. These results suggest the involvement of endothelial-mediated injury and inflammation as the critical mechanisms in cryoinjury and the use of vascular-targeting molecules such as TNF-alpha to enhance tumor killing and achieve the clinical goal of complete cell death within an iceball.

Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice.

Transgenic sickle mice expressing betaS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-kappaB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation-induced stasis, leukocyte-endothelium interactions, and NF-kappaB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-kappaB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation-induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice.

Microvascular blood flow and stasis in transgenic sickle mice: utility of a dorsal skin fold chamber for intravital microscopy.

Vascular inflammation, secondary to ischemia-reperfusion injury, may play an essential role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice expressing human alpha and beta(s)/beta(s-Antilles) globin chains. Microvessels in the DSFC were visualized by intravital microscopy at baseline in ambient air and after exposure to hypoxia-reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle mice after hypoxia-reoxygenation but remained constant in normal mice. The mean RBC velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after hypoxia-reoxygenation. None of the venules in normal mice became static at any time during hypoxia-reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation, 11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and 4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous venules in sickle mice remained static (P = 0.01). All of the venules were flowing again after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous venules of sickle mice by tumor necrosis factor alpha (TNF-alpha). Leukocyte rolling flux and firm adhesion, manifestations of vascular inflammation, were significantly higher at baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle (P < 0.01), but not in normal mice, after hypoxia-reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were also seen in subcutaneous venules.