Vascular and metabolic effects of the haem oxygenase-1 inducer haem arginate in subjects with the metabolic syndrome: A translational cross-over study.

This translational randomized and vehicle-controlled cross-over study was performed to assess the impact of haem arginate treatment on haem oxygenase-1 induction, endothelial function and insulin sensitivity in subjects with the metabolic syndrome (n = 14). Both treatment periods consisted of 5 days. Haem arginate or vehicle (l-arginine) was administered intravenously on Days 1 and 3. Forearm blood flow in response to acetylcholine and nitroglycerine was measured by venous occlusion plethysmography (Day 3), insulin sensitivity by a hyperinsulinaemic clamp procedure (Day 5). Haem arginate did not improve endothelial function or insulin sensitivity but significantly reduced the vasodilator response to nitroglycerine (p < 0.01). These negative findings are in contrast to the preclinical data, which may be due to short duration of therapy and limited haem oxygenase-1 induction as well as interference by markedly elevated plasma haem levels observed after haem arginate treatment (p < 0.01). Future studies should pay attention to the delicate balance between sufficient dosing and timely normalization of plasma haem levels.

Erythropoietin Attenuates Pulmonary Vascular Remodeling in Experimental Pulmonary Arterial Hypertension through Interplay between Endothelial Progenitor Cells and Heme Oxygenase.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease with a high mortality, characterized by typical angio-proliferative lesions. Erythropoietin (EPO) attenuates pulmonary vascular remodeling in PAH. We postulated that EPO acts through mobilization of endothelial progenitor cells (EPCs) and activation of the cytoprotective enzyme heme oxygenase-1 (HO-1). METHODS: Rats with flow-associated PAH, resembling pediatric PAH, were treated with HO-1 inducer EPO in the presence or absence of the selective HO-activity inhibitor tin-mesoporphyrin (SnMP). HO activity, circulating EPCs and pulmonary vascular lesions were assessed after 3 weeks. RESULTS: In PAH rats, circulating EPCs were decreased and HO activity was increased compared to control. EPO treatment restored circulating EPCs and improved pulmonary vascular remodeling, as shown by a reduced wall thickness and occlusion rate of the intra-acinar vessels. Inhibition of HO activity with SnMP aggravated PAH. Moreover, SnMP treatment abrogated EPO-induced amelioration of pulmonary vascular remodeling, while surprisingly further increasing circulating EPCs as compared with EPO alone. CONCLUSION: In experimental PAH, EPO treatment restored the number of circulating EPCs to control level, improved pulmonary vascular remodeling, and showed important interplay with HO activity. Inhibition of increased HO activity in PAH rats exacerbated progression of pulmonary vascular remodeling, despite the presence of restored number of circulating EPCs. We suggest that both EPO-induced HO-1 and EPCs are promising targets to ameliorate the pulmonary vasculature in PAH.

Cytoprotective responses in HaCaT keratinocytes exposed to high doses of curcumin.

Wound healing is a complex process that involves the well-coordinated interactions of different cell types. Topical application of high doses of curcumin, a plant-derived polyphenol, enhances both normal and diabetic cutaneous wound healing in rodents. For optimal tissue repair interactions between epidermal keratinocytes and dermal fibroblasts are essential. We previously demonstrated that curcumin increased reactive oxygen species (ROS) formation and apoptosis in dermal fibroblasts, which could be prevented by pre-induction of the cytoprotective enzyme heme oxygenase (HO)-1. To better understand the effects of curcumin on wound repair, we now assessed the effects of high doses of curcumin on the survival of HaCaT keratinocytes and the role of the HO system. We exposed HaCaT keratinocytes to curcumin in the presence or absence of the HO-1 inducers heme (FePP) and cobalt protoporphyrin (CoPP). We then assessed cell survival, ROS formation, and caspase activation. Curcumin induced caspase-dependent apoptosis in HaCaT keratinocytes via a ROS-dependent mechanism. Both FePP and CoPP induced HO-1 expression, but only FePP protected against curcumin-induced ROS formation and caspase-mediated apoptosis. In the presence of curcumin, FePP but not CoPP induced the expression of the iron scavenger ferritin. Together, our data show that the induction of ferritin, but not HO, protects HaCaT keratinocytes against cytotoxic doses of curcumin. The differential response of fibroblasts and keratinocytes to high curcumin doses may provide the basis for improving curcumin-based wound healing therapies.

Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts.

Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-ß1 play a key role in these processes. The persistence of (myo)fibroblasts and their excessive ECM production and continuous wound contraction have been linked to pathological scarring. The identification of compounds reducing myofibroblast survival and function may thus offer promising therapeutic strategies to optimize impaired wound healing. The plant-derived polyphenol curcumin has shown promising results as a wound healing therapeutic in vivo; however, the exact mechanism is still unclear. In vitro, curcumin induces apoptosis in various cell types via a reactive oxygen species (ROS)-dependent mechanism. Here we treated human dermal fibroblasts with TGF-ß1 to induce myofibroblast differentiation, and compared the responses of fibroblasts and myofibroblasts to 25 µM curcumin. Curcumin induced caspase-independent apoptosis in both fibroblasts and myofibroblasts in a ROS-dependent manner. Oxidative stress leads to the induction of several antioxidant systems to regain cellular homeostasis. We detected stress-induced induction of heme oxygenase (HO)-1 in fibroblasts but not in myofibroblasts following curcumin exposure. Instead, myofibroblasts expressed higher levels of heat shock protein (HSP)72 compared to fibroblasts in response to curcumin, suggesting that TGF-ß1 treatment alters the stress-responses of the cells. However, we did not detect any differences in curcumin toxicity between the two populations. The differential stress responses in fibroblasts and myofibroblasts may open new therapeutic approaches to reduce myofibroblasts and scarring.

Delayed cutaneous wound closure in HO-2 deficient mice despite normal HO-1 expression.

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO-1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO-2 deficient mice is impaired with exorbitant inflammation and absence of HO-1 expression. This study addresses the role of HO-2 in cutaneous excisional wound healing using HO-2 knockout (KO) mice. Here, we show that HO-2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO-2 KO mice compared to WT controls. Surprisingly, wound closure in HO-2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO-1 induction in HO-2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C-X-C) ligand-11 (CXCL-11) in wounds of HO-2 KO mice. Abnormal regulation of CXCL-11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL-11 expression in HO-2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.

Heme Oxygenase-1 and breast cancer resistance protein protect against heme-induced toxicity.

Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating oxidative and inflammatory injury. Protective mechanisms against free heme are therefore pivotal for cellular survival. We postulated that overexpression of Heme Oxygenase-1 (HO-1) and Breast Cancer Resistance Protein (BCRP) would protect against heme-induced cytotoxicity. HO-1 is a heme-degrading enzyme generating carbon monoxide, iron, and biliverdin/bilirubin, while BCRP is a heme efflux transporter. Human embryonic kidney cells were transduced using a baculovirus system as a novel strategy to efficiently overexpress HO-1 and BCRP. Exposing cells to heme resulted in a dose-dependent increase in reactive oxygen species formation, DNA damage and cell death. Heme-induced cell death was significantly attenuated when cells overexpressed HO-1, BCRP, or both. The protective effects of HO-1 overexpression were most pronounced, while co-treatment with the HO-activity inhibitor tin mesoporphyrin reversed these protective effects. Also cells treated with the anti-oxidants N-acetylcysteine or HO-effector molecule bilirubin showed protection against heme insults, which may explain the increased protection by HO-1 compared to BCRP. In conclusion, both HO-1 and BCRP protect against heme-induced toxicity and may thus form novel therapeutic targets for heme-mediated pathologies.

Involvement of VDAC, Bax and ceramides in the efflux of AIF from mitochondria during curcumin-induced apoptosis.

BACKGROUND: We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process. PRINCIPAL FINDINGS: Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3beta (GSK-3beta) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion. CONCLUSIONS: Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.

DNA methylation immediately adjacent to active histone marking does not silence transcription.

Active promoters generally contain histone H3/H4 hyperacetylation and tri-methylation at H3 lysine 4, whereas repressed promoters are associated with DNA methylation. Here we show that the repressed erythroid-specific carbonic anhydrase II (CAII) promoter has active histone modifications localized around the transcription start, while high levels of CpG methylation are present directly upstream from these active marks. Despite the presence of active histone modifications, the repressed promoter requires hormone-induced activation for efficient preinitiation complex assembly. Transient and positional changes in histone H3/H4 acetylation and local changes in nucleosome density are evident during activation, but the bipartite epigenetic code is stably maintained. Our results suggest that active histone modifications may prevent spreading of CpG methylation towards the promoter and show that repressive DNA methylation immediately adjacent to a promoter does not necessarily repress transcription.

Histone modification patterns associated with the human X chromosome.

X inactivation is associated with chromosome-wide establishment of inactive chromatin. Although this is classically regarded as facultative heterochromatin that is uniform in nature, the exact distribution of associated epigenetic marks is not well defined. Here we have analysed histone modifications in human somatic cells within two selected regions of the X chromosome. Intergenic, coding and promoter regions are segregated into differentially marked chromatin. H3K27me3 is most prominent in intergenic and silenced coding regions, but is associated with some active coding regions as well. Histone H3/H4 acetylation and H3K4me3 are locally enriched at promoter regions but do not necessarily mark continuing transcription. Remarkably, H3K9me3 is predominant in coding regions of active genes, a phenomenon that is not restricted to the X chromosome. These results argue against the exclusiveness of individual marks to heterochromatin or euchromatin, but rather suggest that composite patterns of interdependent or mutually exclusive modifications together signal the gene expression status.