Anna Grochot-Przeczek

Stromal cell–derived factor 1 promotes angiogenesis via a heme oxygenase 1–dependent mechanism

Stromal cell–derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C ζ–dependent and vascular endothelial growth factor–independent mechanism. SDF-1–induced endothelial tube formation and migration was impaired in HO-1–deficient cells. Aortic rings from HO-1−/− mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1−/− cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1–deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1–mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.

Heme Oxygenase-1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.

Haem oxygenase-1: non-canonical roles in physiology and pathology

HO-1 (haem oxygenase-1) is a ubiquitously expressed inducible enzyme degrading haem to CO, biliverdin and Fe(2+). Its activation reduces oxidative stress in cells and inhibits inflammation, both due to removal of haem and because of the biological activity of HO-1 products. CO may act similarly to NO, activating soluble guanylate cyclase and elevating cGMP production. It inhibits platelet aggregation, reduces leucocyte adhesion, decreases apoptosis and lowers the production of some pro-inflammatory cytokines. Biliverdin is converted into bilirubin by biliverdin reductase, and both compounds are potent antioxidants, free radical scavengers and inhibitors of the complement cascade. Iron ions can be potentially toxic, increasing the generation of hydroxyl radicals, but simultaneous induction of ferritin and activation of the Fe-ATPase iron transporter protects cells from oxidative stress. Importantly, basal and induced expression of HO-1 is very variable in the human population because of the highly polymorphic (GT)n fragment in the promoter, which may have clinical relevance. The recognized roles of HO-1 are far beyond cytoprotection. The enzyme is important in the regulation of cell proliferation, differentiation and apoptosis. Its activity improves neovascularization, attenuates inflammation and modulates the immune response, thereby influencing carcinogenesis, wound healing, transplant survival and the progression of cardiovascular diseases. Recent results indicate that HO-1 may also act through the regulation of microRNAs, which suggests a much broader involvement of HO-1 in the modulation of cell functions and offers a potential explanation for some well-known activities whose mechanism has hitherto been unclear.

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

Heme Oxygenase-1 Inhibits Myoblast Differentiation by Targeting Myomirs

AIMS Heme oxygenase-1 (HMOX1) is a cytoprotective enzyme degrading heme to biliverdin, iron ions, and carbon monoxide, whose expression is induced in response to oxidative stress. Its overexpression has been suggested as a strategy improving survival of transplanted muscle precursors. RESULTS Here we demonstrated that HMOX1 inhibits differentiation of myoblasts and modulates miRNA processing: downregulates Lin28 and DGCR8, lowers the total pool of cellular miRNAs, and specifically blocks induction of myomirs. Genetic or pharmacological activation of HMOX1 in C2C12 cells reduces the abundance of miR-1, miR-133a, miR-133b, and miR-206, which is accompanied by augmented production of SDF-1 and miR-146a, decreased expression of MyoD, myogenin, and myosin, and disturbed formation of myotubes. Similar relationships between HMOX1 and myomirs were demonstrated in murine primary satellite cells isolated from skeletal muscles of HMOX1(+/+), HMOX1(+/-), and HMOX1(-/-) mice or in human rhabdomyosarcoma cell lines. Inhibition of myogenic development is independent of antioxidative properties of HMOX1. Instead it is mediated by CO-dependent inhibition of c/EBPδ binding to myoD promoter, can be imitated by SDF-1, and partially reversed by enforced expression of miR-133b and miR-206. Control C2C12 myoblasts injected to gastrocnemius muscles of NOD-SCID mice contribute to formation of muscle fibers. In contrast, HMOX1 overexpressing C2C12 myoblasts form fast growing, hyperplastic tumors, infiltrating the surrounding tissues, and disseminating to the lungs. INNOVATION We evidenced for the first time that HMOX1 inhibits differentiation of myoblasts, affects the miRNA processing enzymes, and modulates the miRNA transcriptome. CONCLUSION HMOX1 improves the survival of myoblasts, but concurrently through regulation of myomirs, may act similarly to oncogenes, increasing the risk of hyperplastic growth of myogenic precursors.

Therapeutic angiogenesis for revascularization in peripheral artery disease.

Therapeutic angiogenesis for peripheral artery disease (PAD), achieved by gene and cell therapy, has recently raised a great deal of hope for patients who cannot undergo standard revascularizing treatment. Although pre-clinical studies gave very promising data, still clinical trials of gene therapy have not provided satisfactory results. On the other hand, cell therapy approach, despite several limitations, demonstrated more beneficial effects but initial clinical studies must be constantly validated by larger randomized, multi-center, double-blinded, placebo-controlled trials. This review focuses on previous and recent gene and cell therapy studies for limb ischemia, including both experimental and clinical research, and summarizes some important papers published in this field. Moreover, it provides a short comment on combined gene and cell therapy approach on the example of heme oxygenase-1 overexpressing cells with therapeutic properties.

Role of Heme Oxygenase-1 in Human Endothelial Cells Lesson From the Promoter Allelic Variants

Objective—Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. Methods and Results—On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT ≤23), medium (moderately active, GT=24 to 28), and long (least active, GT ≥29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J2, hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor–induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1&bgr;, interleukin-6, and soluble intercellular adhesion molecule-1. Conclusion—The (GT)n promoter polymorphism significantly modulates a cytoprotective, proangiogenic, and antiinflammatory function of HO-1 in human endothelium.

Cellular and molecular mechanisms of inflammation‐induced angiogenesis

Blood vessel formation is a fundamental process for the development of organism and tissue regeneration. Of importance, angiogenesis occurring during postnatal development is usually connected with inflammation. Here, we review how molecular and cellular mechanisms underlying inflammatory reactions regulate angiogenesis. Inflamed tissues are characterized by hypoxic conditions and immune cell infiltration. In this review, we describe an interplay of hypoxia‐inducible factors (HIFs), HIF1 and HIF2, as well as NF‐κB and nitric oxide in the regulation of angiogenesis. The mobilization of macrophages and the differential role of M1 and M2 macrophage subsets in angiogenesis are also discussed. Next, we present the current knowledge about microRNA regulation of inflammation in the context of new blood vessel formation. Finally, we describe how the mechanisms involved in inflammation influence tumor angiogenesis. We underlay and discuss the role of NF‐E2‐related factor 2/heme oxygenase‐1 pathway as crucial in the regulation of inflammation‐induced angiogenesis.

Nrf2 Regulates Angiogenesis: Effect on Endothelial Cells, Bone Marrow-Derived Proangiogenic Cells and Hind Limb Ischemia

AIMS Nuclear factor E2-related factor 2 (Nrf2), a key cytoprotective transcription factor, regulates also proangiogenic mediators, interleukin-8 and heme oxygenase-1 (HO-1). However, hitherto its role in blood vessel formation was modestly examined. Particularly, although Nrf2 was shown to affect hematopoietic stem cells, it was not tested in bone marrow-derived proangiogenic cells (PACs). Here we investigated angiogenic properties of Nrf2 in PACs, endothelial cells, and inflammation-related revascularization. RESULTS Treatment of endothelial cells with angiogenic cytokines increased nuclear localization of Nrf2 and induced expression of HO-1. Nrf2 activation stimulated a tube network formation, while its inhibition decreased angiogenic response of human endothelial cells, the latter effect reversed by overexpression of HO-1. Moreover, lack of Nrf2 attenuated survival, proliferation, migration, and angiogenic potential of murine PACs and affected angiogenic transcriptome in vitro. Additionally, angiogenic capacity of PAC Nrf2(-/-) in in vivo Matrigel assay and PAC mobilization in response to hind limb ischemia of Nrf2(-/-) mice were impaired. Despite that, restoration of blood flow in Nrf2-deficient ischemic muscles was better and accompanied by increased oxidative stress and inflammatory response. Accordingly, the anti-inflammatory agent etodolac tended to diminish blood flow in the Nrf2(-/-) mice. INNOVATION Identification of a novel role of Nrf2 in angiogenic signaling of endothelial cells and PACs. CONCLUSION Nrf2 contributes to angiogenic potential of both endothelial cells and PACs; however, its deficiency increases muscle blood flow under tissue ischemia. This might suggest a proangiogenic role of inflammation in the absence of Nrf2 in vivo, concomitantly undermining the role of PACs in such conditions.

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1+/+, HO-1+/−, and HO-1−/− mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1−/− and HO-1+/− than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1−/− lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.