Identification of heat shock protein 32 (Hsp32) as a novel survival factor and therapeutic target in neoplastic mast cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 661-669 ◽  
Author(s):  
Rudin Kondo ◽  
Karoline V. Gleixner ◽  
Matthias Mayerhofer ◽  
Anja Vales ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Therapeutic Target ◽  
Systemic Mastocytosis ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Survival Factor ◽  
Myeloid Neoplasm ◽  
Kit D816v

AbstractSystemic mastocytosis (SM) is a myeloid neoplasm characterized by increased survival and accumulation of neoplastic mast cells (MCs). In most patients, the D816V-mutated variant of KIT is detectable. We report here that heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a novel KIT-inducible survival factor in neoplastic MCs. As assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and Western blotting, the KIT D816V+ MC line HMC-1.2 as well as highly enriched primary neoplastic MCs were found to express Hsp32 mRNA and the Hsp32 protein. Moreover, KIT D816V and stem cell factor (SCF)–activated wild-type KIT were found to induce Hsp32 promoter activity, expression of Hsp32 mRNA, and expression of the Hsp32 protein in Ba/F3 cells. Correspondingly, the KIT D816V-targeting drug PKC412 decreased the expression of Hsp32 as well as proliferation/survival in neoplastic MCs. The inhibitory effects of PKC412 on the survival of HMC-1.2 cells were counteracted by the HO-1 inductor hemin or lentiviral-transduced HO-1. Moreover, 2 Hsp32-targeting drugs, pegylated zinc protoporphyrin (PEG-ZnPP) and styrene maleic acid copolymer micelle-encapsulated ZnPP (SMA-ZnPP), were found to inhibit proliferation and to induce apoptosis in neoplastic MCs. Furthermore, both drugs were found to cooperate with PKC412 in producing growth inhibition. Together, these data show that Hsp32 is an important survival factor and interesting new therapeutic target in neoplastic MCs.

Heme Oxygenase-1 (HO-1): A Novel KIT D816V-Dependent Target in Neoplastic Human Mast Cells (HMC-1).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3521-3521 ◽  
Author(s):  
Rudin Kondo ◽  
Matthias Mayerhofer ◽  
Karoline Gleixner ◽  
Anja Vales ◽  
Maria-Theresa Krauth ◽  
...  
Keyword(s):  
Mast Cells ◽  
Heme Oxygenase ◽  
Kinase Inhibitor ◽  
Northern Blotting ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Survival Factor ◽  
Kit D816v

Abstract Systemic mastoyctosis is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC). Most patients express the D816V-mutated variant of c-KIT, which mediates resistance against most available tyrosine kinase inhibitors. Therefore, current research is focusing on novel targets in MC. We analyzed expression and function of the survival factor heme oxygenase-1 (HO-1) in neoplastic MC. As assessed by Northern blotting and RT-PCR, the human MC line HMC-1 that exhibits KIT D816V, was found to express HO-1 mRNA. Expression of the HO-1 protein was demonstrable by immunocytochemistry and Western blotting. To examine the role of mutated KIT in expression of HO-1, Ba/F3 cells with doxycycline-inducible expression of c-KIT D816V were employed. In these experiments, KIT D816V was found to induce HO-1 promoter activity and expression of HO-1 mRNA and HO-1 protein in these cells. Moreover, the KIT-D816V-targeting compound PKC412 was found to downregulate expression of HO-1 in HMC-1 cells. The KIT D816V-induced upregulation of HO-1 in Ba/F3 cells was completely blocked by a combination of LY294002 (PI3-kinase inhibitor) and PD89059 (MEK inhibitor), but not by single agents, suggesting involvement of multiple signaling pathways. We next examined whether targeting of HO-1 is associated with decreased survival. As assessed by 3H-thymidine uptake, the pegylated HO-1 inhibitor zinc-protoporphyrin (PEG-ZnPP) reduced proliferation of HMC-1 cells in a dose-dependent manner (IC50: 5 μM). The PEG-ZnPP-induced inhibition of growth was found to be associated with induction of apoptosis (control: 1±0.6 vs PEG-ZnPP, 5 μM: 55±5% apoptotic cells, p<0.05). The HO-1 inductor hemin (10 μM) increased the expression of HO-1 in HMC-1 cells and partly rescued these cells from PKC412-induced growth-inhibition (PKC412, 1 μM: 39±8% vs PKC412 + hemin: 23±11%). Finally, PKC412 and PEG-ZnPP were found to produce cooperative (additive) growth-inhibitory effects on HMC-1 cells. In summary, our data show that HO-1 is a novel survival factor and interesting target in neoplastic human MC exhibiting the D816V-mutated variant of KIT.

Delineation of a KIT-Independent Oncogenic Pathway in Neoplastic Mast Cells That Involves Lyn and Btk, and Can Be Disrupted by the KIT/Lyn/Btk-Targeting Drug Dasatinib

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1541-1541 ◽  
Author(s):  
Karoline V. Gleixner ◽  
Matthias Mayerhofer ◽  
Uwe Rix ◽  
Gregor Hoermann ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Potent Inhibitor ◽  
Systemic Mastocytosis ◽  
Synergistic Effects ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Growth And Survival ◽  
Myeloid Neoplasm ◽  
Oncogenic Pathway ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloid neoplasm characterized by increased growth and survival of neoplastic mast cells (MC). Aggressive SM (ASM) and MC leukemia (MCL) are advanced disease variants that usually are drug-resistant and have an unfavorable prognosis. In most patients, the D816V-mutated ′oncogenic′ variant of KIT is detectable. However, the mutant is also detectable in patients with indolent SM exhibiting a normal life-expectancy, and therefore is not considered to represent a fully transforming oncoprotein. This assumption is also supported by studies in Ba/F3 cells, and whether KIT D816V-targeting drugs are able to induce long-term remission in ASM or MCL, remains to be seen. Therefore, it has been hypothesized that in addition to KIT, other pro-oncogenic molecules and signaling pathways play a role in malignant transformation/progression in SM. We here describe a novel KIT D816V-independent oncogenic pathway in neoplastic MC that involves Lyn and Bruton’s tyrosine kinase (Btk). Western blotting and immunostaining revealed that neoplastic MC display the Btk- and Lyn protein. Both molecules were found to be constitutively phosphorylated in primary neoplastic MC and in the MC leukemia cell line HMC-1. Lyn/Btk-activation was not only detectable in KIT D816V-positive HMC-1.2 cells, but also in the KIT D816V-negative HMC-1.1 subclone. In studies employing Ba/F3 cells with doxycycline-inducible expression of KIT, we were able to show that KIT D816V induces activation of STAT5 and Akt, but does not induce activation of Btk. Correspondingly, pharmacologic deactivation/dephosphorylation of KIT in HMC-1 cells by midostaurin (PKC412) (Novartis, Basel, Switzerland) was not accompanied by a decrease in phosphorylation of Lyn or Btk. The functional significance of Btk expression/activation in neoplastic MC could be demonstrated by a Btk-specific siRNA that reduced the proliferation and survival in HMC-1 cells, and was found to cooperate with midostaurin in producing growth inhibition. In consecutive experiments, we identified the Src/Abl kinase-targeting drug dasatinib (BMS, Princeton, NJ) as a potent inhibitor of Lyn/Btk activation in neoplastic MC. In particular, dasatinib (1 μM) was found to block Lyn and Btk activity in HMC-1.1 cells as well as in HMC-1.2 cells, and corresponding results were obtained with primary neoplastic MC. Finally, as assessed by a chemical proteomics approach, we were able to show that dasatinib directly binds to Btk and Lyn in neoplastic MC. In summary, our data show that a KIT-independent Lyn/Btk-driven signaling pathway contributes to growth and survival of neoplastic MC, and possibly to disease progression in SM. Our study also identifies dasatinib as a potent inhibitor of the Lyn/Btk pathway, which may have clinical implications and may explain some of the synergistic effects obtained with combinations of dasatinib and other KIT-targeting TK inhibitors in neoplastic MC.

Heme Oxygenase-1 (HO-1)/Heat Shock Protein 32 (Hsp32) as a Novel Survival Factor and Target in AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1901-1901 ◽  
Author(s):  
Michael Kneidinger ◽  
Karoline V. Gleixner ◽  
Rudin Kondo ◽  
Puchit Samorapoompichit ◽  
Anja Vales ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Heme Oxygenase ◽  
Leukemic Cells ◽  
Heme Oxygenase 1 ◽  
Inflammatory Reactions ◽  
Survival Factor ◽  
Dependent Inhibition

Abstract Heme oxygenase 1 (HO-1), also known as heat shock protein 32 (Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. Recent data suggest that HO-1/Hsp32 is also expressed in neoplastic cells in various malignancies. In the present study, we provide evidence that HO-1 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML, n=17) and in various AML cell lines such as HL60, KG1, KG1a, and U937. Expression of HO-1 mRNA was demonstrable by RT-PCR, and the HO-1 protein by immunocytochemistry and Western blotting. In addition, we were able to demonstrate expression of HO-1 mRNA and of HO-1 protein in the CD34+/CD38− progenitor/stem cell fraction in the leukemic clone in patients with AML. The HO-1 inductor hemin (10 μM) was found to promote expression of HO-1 in AML cells. Incubation with the HO-1-targeting drugs pegylated zink protoporphyrin (PEG-ZnPP) or styrene maleic acid-conjugated ZnPP (SMA-ZnPP), resulted in a dose-dependent inhibition of growth of leukemic cells at pharmacologic concentrations (IC50: 5–20 μM for cell lines and primary AML cells). The SMA-ZnPP-induced growth-inhibition of AML cells were found to be associated with induction of apoptosis as evidenced by light microscopy, electron microscopy, and by a Tunel assay. In consecutive experiments, combination experiments were performed using SMA-ZnPP and AML cell lines. In these experiments, SMA-ZnPP was found to synergize with cytarabine in producing growth inhibition in all AML cell lines tested. In summary, these data show that HO-1/Hsp32 is a novel survival factor and interesting target in AML. The clinical significance of this observation remains to be determined in forthcoming trials.

Identification of MCL1 as a novel target in neoplastic mast cells in systemic mastocytosis: inhibition of mast cell survival by MCL1 antisense oligonucleotides and synergism with PKC412

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 3031-3041 ◽  
Author(s):  
Karl J. Aichberger ◽  
Matthias Mayerhofer ◽  
Karoline V. Gleixner ◽  
Maria-Theresa Krauth ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Antisense Oligonucleotides ◽  
Kinase Inhibitors ◽  
Systemic Mastocytosis ◽  
Synergistic Effects ◽  
Kit Mutation ◽  
Myeloid Neoplasm ◽  
Novel Target ◽  
Kit D816v

Abstract MCL-1 is a Bcl-2 family member that has been described as antiapoptotic in various myeloid neoplasms. Therefore, MCL-1 has been suggested as a potential new therapeutic target. Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In the present study, we examined the expression and functional role of MCL-1 in neoplastic MCs and sought to determine whether MCL-1 could serve as a target in SM. As assessed by RT-PCR and immunohistochemical examination, primary neoplastic MCs expressed MCL-1 mRNA and the MCL-1 protein in all SM patients examined. Moreover, MCL-1 was detectable in both subclones of the MC line HMC-1—HMC-1.1 cells, which lack the SM-related KIT mutation D816V, and HMC-1.2 cells, which carry KIT D816V. Exposure of HMC-1.1 cells or HMC-1.2 cells to MCL-1–specific antisense oligonucleotides (ASOs) or MCL-1–specific siRNA resulted in reduced survival and increased apoptosis compared with untreated cells. Moreover, MCL-1 ASOs were found to cooperate with various tyrosine kinase inhibitors in producing growth inhibition in neoplastic MCs, with synergistic effects observed with PKC412, AMN107, and imatinib in HMC-1.1 cells and with PKC412 in HMC-1.2 cells. Together, these data show that MCL-1 is a novel survival factor and an attractive target in neoplastic MCs.

Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs

Blood ◽  
2009 ◽  
Vol 114 (26) ◽  
pp. 5342-5351 ◽  
Author(s):  
Karl J. Aichberger ◽  
Karoline V. Gleixner ◽  
Irina Mirkina ◽  
Sabine Cerny-Reiterer ◽  
Barbara Peter ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tumor Suppressor ◽  
Systemic Mastocytosis ◽  
Leukemia Cell ◽  
Myeloid Neoplasm ◽  
Myeloid Neoplasms ◽  
Leukemia Cell Lines ◽  
Survival And Growth ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816–induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.

scholarly journals New Insights into the Pathogenesis of Systemic Mastocytosis

2021 ◽  
Vol 22 (9) ◽  
pp. 4900
Author(s):  
Zhixiong Li
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Systemic Mastocytosis ◽  
Treatment Strategies ◽  
The United States ◽  
European Medicines Agency ◽  
Myeloid Neoplasm ◽  
Organ Systems ◽  
United States Food ◽  
Kit D816v

Mastocytosis is a type of myeloid neoplasm characterized by the clonal, neoplastic proliferation of morphologically and immunophenotypically abnormal mast cells that infiltrate one or more organ systems. Systemic mastocytosis (SM) is a more aggressive variant of mastocytosis with extracutaneous involvement, which might be associated with multi-organ dysfunction or failure and shortened survival. Over 80% of patients with SM carry the KIT D816V mutation. However, the KIT D816V mutation serves as a weak oncogene and appears to be a late event in the pathogenesis of mastocytosis. The management of SM is highly individualized and was largely palliative for patients without a targeted form of therapy in past decades. Targeted therapy with midostaurin, a multiple kinase inhibitor that inhibits KIT, has demonstrated efficacy in patients with advanced SM. This led to the recent approval of midostaurin by the United States Food and Drug Administration and European Medicines Agency. However, the overall survival of patients treated with midostaurin remains unsatisfactory. The identification of genetic and epigenetic alterations and understanding their interactions and the molecular mechanisms involved in mastocytosis is necessary to develop rationally targeted therapeutic strategies. This review briefly summarizes recent developments in the understanding of SM pathogenesis and potential treatment strategies for patients with SM.

PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated variant of KIT: comparison with AMN107, imatinib, and cladribine (2CdA) and evaluation of cooperative drug effects

Blood ◽  
2006 ◽  
Vol 107 (2) ◽  
pp. 752-759 ◽  
Author(s):  
Karoline V. Gleixner ◽  
Matthias Mayerhofer ◽  
Karl J. Aichberger ◽  
Sophia Derdak ◽  
Karoline Sonneck ◽  
...  
Keyword(s):  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Drug Effects ◽  
Kit Mutation ◽  
Growth Inhibitory ◽  
Ic50 Values ◽  
Tk Inhibitors ◽  
Mast Cell Leukemia ◽  
Kit D816v

AbstractIn most patients with systemic mastocytosis (SM), including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V. KIT D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive drug target. However, imatinib and most other TK inhibitors fail to block the TK activity of KIT D816V. We show that the novel TK-targeting drugs PKC412 and AMN107 counteract TK activity of D816V KIT and inhibit the growth of Ba/F3 cells with doxycycline-inducible expression of KIT D816V as well as the growth of primary neoplastic mast cells and HMC-1 cells harboring this KIT mutation. PKC412 was a superior agent with median inhibitory concentration (IC50) values of 50 to 250 nM without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited more potent effects in KIT D816V- HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or D816V-mutated KIT. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with induction of apoptosis and down-regulation of CD2 and CD63. PKC412 was found to cooperate with AMN107, imatinib, and cladribine (2CdA) in producing growth inhibition in HMC-1, but synergistic drug interactions were observed only in cells lacking KIT D816V. Together, PKC412 and AMN107 represent promising novel agents for targeted therapy of SM. (Blood. 2006;107: 752-759)

Antiapoptotic function of Bcl-2 in mast cells is dependent on its association with heat shock protein 90β

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1413-1420 ◽  
Author(s):  
Cellina Cohen-Saidon ◽  
Irit Carmi ◽  
Avishai Keren ◽  
Ehud Razin
Keyword(s):  
Mast Cell ◽  
Rna Interference ◽  
Mast Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Caspase 3 ◽  
Caspase 7 ◽  
Antiapoptotic Activity ◽  
Novel Strategy ◽  
First Time

In the present study, we demonstrated that the antiapoptotic function of Bcl-2 in mast cells is significantly dependent on its association with the heat shock protein 90β (Hsp90β). Dissociation of these 2 proteins inhibits the antiapoptotic activity of Bcl-2 by initiating the release of cytochrome c from mitochondria into cytosol and increasing the activity of caspase 3 and caspase 7, resulting in mast-cell apoptosis. The antiapoptotic activity of Bcl-2 was greatly affected by knocking-out specifically Hsp90β using the RNA interference approach. Thus, for the first time, it has been shown that Hsp90β might modulate the antiapoptotic activity of Bcl-2 at least in mast cells. These findings could have implications for a novel strategy of regulating apoptosis in patients with mastocytosis and other mast cell–associated diseases.

Vascular Endothelial Growth Factors and Angiopoietins As New Players in Mastocytosis

2021 ◽  
Author(s):  
Simone Marcella ◽  
Angelica Petraroli ◽  
Mariantonia Braile ◽  
Roberta Parente ◽  
Anne Lise Ferrara ◽  
...  
Keyword(s):  
Growth Factors ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Vascular Endothelial Growth Factors ◽  
Vascular Endothelial ◽  
Serum Concentrations ◽  
Kit Receptor ◽  
Clinical Variants ◽  
Endothelial Growth Factors ◽  
Kit D816v

Abstract Mastocytosis is a disorder characterized by the abnormal proliferation and/or accumulation of mast cells in different organs. More than 90% of patients with systemic mastocytosis have a gain-of-function mutation in codon 816 of the KIT receptor on mast cells (MCs). The symptoms of mastocytosis patients are related to the MC-derived mediators that exert local and distant effects. MCs produce angiogenic and lymphangiogenic factors, including vascular endothelial growth factors (VEGFs) and angiopoietins (ANGPTs). Serum concentrations of VEGF-A, VEGF-C, VEGF-D, ANGPT1 and ANGPT2 were determined in 64 mastocytosis patients and 64 healthy controls. Intracellular concentrations and spontaneous release of these mediators were evaluated in the mast cell lines ROSAKIT WT and ROSA KIT D816V and in human lung mast cells (HLMCs). VEGF-A, ANGPT1, ANGPT2 and VEGF-C concentrations were higher in mastocytosis patients compared to controls. The VEGF-A, ANGPT2 and VEGF-C concentrations were correlated with the symptom severity. ANGPT1 concentrations were increased in all patients compared to controls. ANGPT2 levels were correlated with severity of clinical variants and with tryptase levels. VEGF-A, ANGPT1 and VEGF-C did not differ between indolent and advanced mastocytosis. ROSAKIT WT, ROSAKIT D816V and HLMCs contained and spontaneously released VEGFs and ANGPTs. Serum concentrations of VEGFs and ANGPTs are altered in mastocytosis patients.

Mechanism of the delay phenomenon: tissue protection is mediated by heme oxygenase-1

2004 ◽  
Vol 287 (5) ◽  
pp. H2332-H2340 ◽  
Author(s):  
Darius Kubulus ◽  
Frank Roesken ◽  
Michaela Amon ◽  
Martin Rücker ◽  
Michael Bauer ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Skin Flap ◽  
Heme Oxygenase 1 ◽  
Flap Necrosis ◽  
Tissue Necrosis ◽  
Chronic Ischemia ◽  
Tissue Protection ◽  
Delay Phenomenon

Induction of the “delay phenomenon” by chronic ischemia is an established clinical procedure, but the mechanisms conferring tissue protection are still incompletely understood. To elucidate the role of heme oxygenase-1 [HO-1 or heat shock protein-32 (HSP-32)] in delay, we examined in the skin-flap model of the ear of the hairless mouse, 1) whether chronic ischemia (delay) is capable to induce expression of HO-1, and 2) whether delay-induced HO-1 affects skin-flap microcirculation and survival by either its carbon monoxide-associated vasodilatory action or its biliverdin-associated anti-oxidative mechanism. Chronic ischemia was induced by transsection of the central feeding vessel of the ear 7 days before flap creation. The flap was finally raised by an incision through four-fifths of the base of the ear. Microcirculatory dysfunction and tissue necrosis were studied with the use of laser Doppler fluxmetry and intravital fluorescence microscopy. HO-1 protein expression was determined with Western blot analysis. Seven days of chronic ischemia (delay) induced a marked expression of HO-1. This was paralleled by a significant improvement ( P < 0.05) of microvascular perfusion and a reduction ( P < 0.05) of flap necrosis when compared with nondelayed controls. Importantly, blockade of HO-1 activity by tin protoporhyrin-IX completely blunted the protection of microcirculation and the improvement of tissue survival. Additional administration of the vitamin E analog trolox after blockade of HO-1 to mimic exclusively the anti-oxidative action of the heat shock protein did not restore the HO-1-associated microcirculatory improvement and only transiently attenuated the manifestation of flap necrosis. Thus our data indicate that the delay-induced protection from tissue necrosis is mediated by HO-1, predominantly through its carbon monoxide-associated action of adequately maintaining nutritive capillary perfusion.

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