Lars Bellner

Heme Oxygenase-1 Induction Attenuates Corneal Inflammation and Accelerates Wound Healing After Epithelial Injury

PURPOSE Heme oxygenase (HO) is considered a fundamental endogenous immunomodulatory, cytoprotective, and anti-inflammatory system. This protective function is primarily ascribed to the inducible HO-1. The authors examined the effect of HO-1 induction on corneal inflammation and wound healing in mice undergoing epithelial injury. METHODS C57BL6 mice were treated with SnCl(2) the day before epithelial injury and once daily thereafter. The corneal epithelium was removed with the use of a corneal rust ring remover in anesthetized mice. Reepithelialization was measured by fluorescein staining. The inflammatory response was examined by histology and was quantified by the myeloperoxidase assay. Inflammatory lipid mediators were detected and quantified by LC/MS/MS-based lipidomic analysis. HO-1 expression was assessed by real-time PCR, and HO activity was determined by measuring HO-dependent carbon monoxide production. RESULTS Epithelial injury caused a time-dependent transient increase in HO-1 expression and HO activity that was significantly amplified by treatment with SnCl(2), resulting in a twofold to threefold increase in mRNA levels and a similar increase in corneal HO activity. Induction of HO-1 was associated with a significant acceleration of wound healing when compared with a vehicle-treated group and with attenuation of the inflammatory response, evidenced by a significant decrease in the number of infiltrating cells and by a significant reduction in the expression and production of proinflammatory lipid mediators and cytokines. CONCLUSIONS Increased expression of HO-1 provides a mechanism that modulates inflammation and promotes wound closure; pharmacologic amplification of this system may constitute a novel strategy to treat corneal inflammation while accelerating wound repair after injury.

Increased heme-oxygenase 1 expression in mesenchymal stem cell-derived adipocytes decreases differentiation and lipid accumulation via upregulation of the canonical Wnt signaling cascade

IntroductionHeme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes.MethodsTo verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining.ResultsDuring adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP.ConclusionsThis is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.

Profile of Lipid and Protein Autacoids in Diabetic Vitreous Correlates with the Progression of Diabetic Retinopathy

OBJECTIVE This study was aimed at obtaining a profile of lipids and proteins with a paracrine function in normal and diabetic vitreous and exploring whether the profile correlates with retinal pathology. RESEARCH DESIGN AND METHODS Vitreous was recovered from 47 individuals undergoing vitreoretinal surgery: 16 had nonproliferative diabetic retinopathy (NPDR), 15 had proliferative diabetic retinopathy, 7 had retinal detachments, and 9 had epiretinal membranes. Protein and lipid autacoid profiles were determined by protein arrays and mass spectrometry–based lipidomics. RESULTS Vitreous lipids included lipoxygenase (LO)- and cytochrome P450 epoxygenase (CYP)-derived eicosanoids. The most prominent LO-derived eicosanoid was 5-hydroxyeicosate traenoic acid (HETE), which demonstrated a diabetes-specific increase (P = 0.027) with the highest increase in NPDR vitreous. Vitreous also contained CYP-derived epoxyeicosatrienoic acids; their levels were higher in nondiabetic than diabetic vitreous (P < 0.05). Among inflammatory, angiogenic, and angiostatic cytokines and chemokines, only vascular endothelial growth factor (VEGF) showed a significant diabetes-specific profile (P < 0.05), although a similar trend was noted for tumor necrosis factor (TNF)-α. Soluble VEGF receptors R1 and R2 were detected in all samples with lowest VEGF-R2 levels (P < 0.05) and higher ratio of VEGF to its receptors in NPDR and PDR vitreous. CONCLUSIONS This study is the first to demonstrate diabetes-specific changes in vitreous lipid autacoids including arachidonate and docosahexanoate-derived metabolites indicating an increase in inflammatory versus anti-inflammatory lipid mediators that correlated with increased levels of inflammatory and angiogenic proteins, further supporting the notion that inflammation plays a role the pathogenesis of this disease.

Heme Oxygenase-2 Deletion Causes Endothelial Cell Activation Marked by Oxidative Stress, Inflammation, and Angiogenesis

In previous studies, we have shown that heme oxygenase (HO)-2 null [HO-2(−/−)] mice exhibit a faulty response to injury; chronic inflammation and massive neovascularization replaced resolution of inflammation and tissue repair. Endothelial cells play an active and essential role in the control of inflammation and the process of angiogenesis. We examined whether HO-2 deletion affects endothelial cell function. Under basal conditions, HO-2(−/−) aortic endothelial cells (mAEC) showed a 3-fold higher expression of vascular endothelial growth factor receptor 1 and a marked angiogenic response compared with wild-type (WT) cells. Compared with WT cells, HO-2(−/−) mAEC showed a 2-fold reduction in HO activity and marked increases in levels of gp91phox/NADPH oxidase isoform, superoxide, nuclear factor κB activation, and expression of inflammatory cytokines, including interleukin (IL)-1α and IL-6. HO-2 deletion transforms endothelial cells from a “normal” to an “activated” phenotype characterized by increases in inflammatory, oxidative, and angiogenic factors. This switch may be the result of reduced HO activity and the associated reduction in the cytoprotective HO products, carbon monoxide and biliverdin/bilirubin, because addition of biliverdin to HO-2(−/−) cells attenuated angiogenesis and reduced superoxide production. This transformation underscores the importance of HO-2 in the regulation of endothelial cell homeostasis.

Astrocyte-Derived CO Is a Diffusible Messenger That Mediates Glutamate-Induced Cerebral Arteriolar Dilation by Activating Smooth Muscle Cell KCa Channels

Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient KCa currents and single KCa channels in myocytes that were in contact with astrocytes, but did not affect KCa channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient KCa currents and KCa channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a KCa channel blocker. These data indicate that an astrocytic signal, notably HO-2–derived CO, is used by glutamate to stimulate arteriole myocyte KCa channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.

Knockdown of Heme Oxygenase-2 Impairs Corneal Epithelial Cell Wound Healing

Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti‐oxidative and anti‐inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO‐2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO‐1 and HO‐2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO‐1 and HO‐2 proteins. Injury elicited a rapid and transient increase in HO‐1 and HO activity; HO‐2 expression was unchanged. Treatment with biliverdin or CORM‐A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM‐A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO‐2 expression, but not HO‐1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO‐2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin. J. Cell. Physiol. 226: 1732–1740, 2011.

Heme oxygenase (HO-1) rescue of adipocyte dysfunction in HO-2 deficient mice via recruitment of epoxyeicosatrienoic acids (EETs) and adiponectin.

Background/Aims: HO-1 and EETs are functionally linked and their interactions influence body weight, insulin sensitivity, and serum levels of inflammatory cytokines in metabolic syndrome phenotype of HO-2 null mice. The HO-2 isozyme is essential for regulating physiological levels of ROS. Recent studies have suggested a potential role of EET in modifying adipocyte differentiation through up-regulation of HO-1-adiponectin-AkT signaling in human mesenchymal stem cells (MSCs). Our aim was to examine the consequences of HO deficiency on MSC-derived adipogenesis in vitro using MSC derived from HO-2 null and WT mice in vivo. Methods: Four-month-old HO-2 null (HO-2-/-) and B6/129SF2/J (WT) mice were divided into three groups (four mice/group): WT, HO-2-/-, and HO-2-/- +CoPP. Adipogenesis was performed on purified MSC-derived adipocytes cultured in adipogenic differentiation media and an EET-agonist was added every 3 days. Results: HO-2 depletion of MSC adipocytes resulted in increased adipogenesis (p<0.01) and increased levels of inflammatory cytokines including (TNF)-alpha (p<0.05), (MCP)-1 (p<0.05), and (IL-1)-beta (p<0.05). These results were accompanied by decreases in HO-1 (p<0.05) and subsequently EET and HO activity (p<0.05). Up-regulation of HO-1 resulted in decreased MSC-derived adipocyte differentiation, decreased production of TNF-alpha and MCP-1 and increased levels of adiponectin (p<0.05). Cyp2J5 (p<0.05), HO-1 (p<0.05), and adiponectin mRNA levels (p<0.05) were also decreased in visceral adipose tissue isolated from HO-2 null compared to WT mice. EET agonist stimulation of MSC adipocytes derived from HO-2 null mice yielded similar results. Conclusion: Increased levels of EET and HO-1 are essential for protection against the adverse effects of adipocyte hypertrophy and the ensuing metabolic syndrome. These results offer a portal into therapeutic approaches for the prevention of the metabolic syndrome.

Epoxyeicosatrienoic acids and heme oxygenase-1 interaction attenuates diabetes and metabolic syndrome complications.

MSCs are considered to be the natural precursors to adipocyte development through the process of adipogenesis. A link has been established between decreased protective effects of EETs or HO-1 and their interaction in metabolic syndrome. Decreases in HO-1 or EET were associated with an increase in adipocyte stem cell differentiation and increased levels of inflammatory cytokines. EET agonist (AKR-I-27-28) inhibited MSC-derived adipocytes and decreased the levels of inflammatory cytokines. We further describe the role of CYP-epoxygenase expression, HO expression, and circulating cytokine levels in an obese mouse, ob/ob(-/-) mouse model. Ex vivo measurements of EET expression within MSCs derived from ob/ob(-/-) showed decreased levels of EETs that were increased by HO induction. This review demonstrates that suppression of HO and EET systems exist in MSCs prior to the development of adipocyte dysfunction. Further, adipocyte dysfunction can be ameliorated by induction of HO-1 and CYP-epoxygenase, i.e. EET.

Biliverdin Rescues the HO-2 Null Mouse Phenotype of Unresolved Chronic Inflammation Following Corneal Epithelial Injury

PURPOSE. The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins by its products biliverdin/bilirubin and carbon monoxide. Corneal injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications, including ulceration and neovascularization. The authors examined whether topically administered biliverdin can counteract the effects of HO deficiency in a corneal epithelial injury model. METHODS. HO-2 null mice were treated with biliverdin 1 hour before epithelial injury and twice a day thereafter. Reepithelialization and neovascularization were assessed by fluorescein staining and vital microscopy, respectively, and were quantified by image analysis. Inflammation was quantified by histology and Gr-1-specific immunofluorescence, and oxidative stress was assessed by DHE fluorescence. RESULTS. Treatment with biliverdin accelerated wound closure, inhibited neovascularization and reduced epithelial defects. It also reduced inflammation, as evidenced by a reduction in the appearance of inflammatory cells and the expression levels of inflammatory and oxidant proteins, including KC and NOXs. CONCLUSIONS. The results clearly show that biliverdin, directly or through its metabolism to bilirubin by biliverdin reductase-the expression of which is increased after injury-rescues the aberrant inflammatory phenotype, further underscoring the importance of the HO system in the cornea for the execution of an ordered inflammatory and reparative response.

PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid.

BACKGROUND/OBJECTIVES Obesity is a risk factor in the development of type 2 diabetes mellitus (DM2), which is associated with increased morbidity and mortality, predominantly as a result of cardiovascular complications. Increased adiposity is a systemic condition characterized by increased oxidative stress (ROS), increased inflammation, inhibition of anti-oxidant genes such as HO-1 and increased degradation of epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs attenuate mitochondrial ROS. We postulate that EETs increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which controls mitochondrial function, oxidative metabolism and induction of HO-1. METHODS Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess functional relationship between EETs, HO-1 and (PGC-1α) using an EET analogue (EET-A) and lentivirus to knock down the PPARGC1A gene. RESULTS EET-A increased PGC-1α and HO-1 in cultured adipocytes and increased the expression of genes involved in thermogenesis and adipocyte browning (UCP1 and PRDM16, respectively). PGC-1α knockdown prevented EET-A-induced HO-1expression, suggesting that PGC-1α is upstream of HO-1. MRI data obtained from fat tissues showed that EET-A administration to mice on a HF diet significantly reduced total body fat content, subcutaneous and visceral fat deposits and reduced the VAT: SAT ratio. Moreover EET-A normalized the VO2 and RQ (VCO2/VO2) in mice fed a HF diet, an effect that was completely prevented in PGC-1α deficient mice. In addition, EET-A increased mitochondrial biogenesis and function as measured by OPA1, MnSOD, Mfn1, Mfn2, and SIRT3, an effect that was inhibited by knockdown of PGC-1α. CONCLUSION Taken together, our findings show that EET-A increased PGC-1α thereby increasing mitochondrial viability, increased fusion potential thereby providing metabolic protection and increased VO2 consumption in HF-induced obesity in mice, thus demonstrating that the EET-mediated increase in HO-1 levels require PGC-1α expression.