Co-administration of angiotensin II and simvastatin triggers kidney injury upon heme oxygenase-1 deficiency.

Kidneys are pivotal organ in iron redistribution and can be severely damaged in the course of hemolysis. In our previous studies, we observed that induction of hypertension with angiotensin II (Ang II) combined with simvastatin administration results in a high mortality rate or the appearance of signs of kidney failure in heme oxygenase-1 knockout (HO-1 KO) mice. Here, we aimed to address the mechanisms underlying this effect, focusing on heme and iron metabolism. We show that HO-1 deficiency leads to iron accumulation in the renal cortex. Higher mortality of Ang II and simvastatin-treated HO-1 KO mice coincides with increased iron accumulation and the upregulation of mucin-1 in the proximal convoluted tubules. In vitro studies showed that mucin-1 hampers heme- and iron-related oxidative stress through the sialic acid residues. In parallel, knock-down of HO-1 induces the glutathione pathway in an NRF2-depedent manner, which likely protects against heme-induced toxicity. To sum up, we showed that heme degradation during heme overload is not solely dependent on HO-1 enzymatic activity, but can be modulated by the glutathione pathway. We also identified mucin-1 as a novel redox regulator. The results suggest that hypertensive patients with less active HMOX1 alleles may be at higher risk of kidney injury after statin treatment.

Slow-cycling murine melanoma cells display plasticity and enhanced tumorigenicity in syngeneic transplantation assay.

Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.

No differences in miRNA expression in the stroma of ERG+ and ERG- prostate cancers.

Prostate cancer (PC) is one of the most common cancers in males. A significant proportion of PCs bear TMPRSS2-ETS translocation and overexpress ERG transcription factor, allowing classification into ERG+ and ERG- groups, which differ in several features including the tumor microenvironment. The aim of the study was to verify whether they differ in expression of the miRNA in the microenvironment. The material consisted of 150 radical prostatectomies. Immunohistochemistry (IHC) for ERG was done using a routine method. FISH for TMPRSS2-ETS translocation was done with a ZytoLight SPEC ERG/TMPRSS2 TriCheck Probe. From each case, a representative section was selected, and tumor and non-tumor were microdissected with the LMD7000 device. RNA was isolated using the RNeasy Mini Kit system (Qiagen) and miRNA libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set for Illumina and their sequencing was performed on the NexSeq 500. Statistical analysis was done with Statistica and R software. When analyzing the expression of miRNAs some differences could be seen, but after correction for multiple comparisons was applied, these were found to be non- significant.

The lack of transcriptionally active Nrf2 triggers colon dysfunction in female mice - The role of estrogens.

BACKGROUND AND AIM: The proper functioning of the gastrointestinal system relies on an intricate crosstalk between a plethora of cell types and signaling pathways. Recently we identified that the lack of NRF2 transcriptional activity (NRF2 tKO) triggers significant colon microscopical alterations, still they do not affect the general functioning of mice. Therefore, in this study, we aimed to address the gender-dependent impact of NRF2 transcriptional deficiency on colon function, and relate them to an established model of inflammatory bowel disease (IBD). METHODS: In the study we subjected 3- and 6-month old mice deficient in IL-10 and NRF2 transcriptional activity and wild-type counterparts to tests assessing colon functionality, and histological analyses. To address the role of estrogens, we attempted to rescue the phenotype by the delivery of 17ß-estradiol through subcutaneous implants. RESULTS: In females, NRF2 transcriptional abrogation, like IL-10 deficiency, triggers a functional and microscopic phenotype, that resembles IBD. The females are significantly more affected by the dysfunctional phenotype, and the functional impairmentdecreases with age. We found that NRF2 transcriptional activity influences 17ß-estradiol level and the estrogen receptors expression and location. Exogenous delivery of 17ß-estradiol normalized colon motility in the NRF2 tKO mice, which is related to enhanced ERß signaling. CONCLUSIONS: Summing up, in this study, we underline that NRF2 transcriptional deficiency or the lack of IL-10 results in pronounced GI functional decline in young females. Mechanistically, we show that the impaired distal colon motility is dependent on ERß signaling. Targeting estrogen signaling seems a promising therapeutic strategy to counteract colonic dysfunction.

Heme Oxygenase-1 Has a Greater Effect on Melanoma Stem Cell Properties Than the Expression of Melanoma-Initiating Cell Markers.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

The biology of hematopoietic stem cells and its clinical implications.

Hematopoietic stem cells (HSCs) give rise to all types of blood cells and self-renew their own population. The regeneration potential of HSCs has already been successfully translated into clinical applications. However, recent studies on the biology of HSCs may further extend their clinical use in future. The roles of HSCs in native hematopoiesis and in transplantation settings may differ. Furthermore, the heterogenic pool of HSCs dynamically changes during aging. These changes also involve the complex interactions of HSCs with the bone marrow niche. Here, we review the opportunities and challenges of these findings to improve the clinical use of HSCs. We describe new methods of HSCs mobilization and conditioning for the transplantation of HSCs. Finally, we highlight the research findings that may lead to overcoming the current limitations of HSC transplantation and broaden the patient group that can benefit from the clinical potential of HSCs.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

The role of heme oxygenase-1 in hematopoietic system and its microenvironment.

Hematopoietic system transports all necessary nutrients to the whole organism and provides the immunological protection. Blood cells have high turnover, therefore, this system must be dynamically controlled and must have broad regeneration potential. In this review, we summarize how this complex system is regulated by the heme oxygenase-1 (HO-1)-an enzyme, which degrades heme to biliverdin, ferrous ion and carbon monoxide. First, we discuss how HO-1 influences hematopoietic stem cells (HSC) self-renewal, aging and differentiation. We also describe a critical role of HO-1 in endothelial cells and mesenchymal stromal cells that constitute the specialized bone marrow niche of HSC. We further discuss the molecular and cellular mechanisms by which HO-1 modulates innate and adaptive immune responses. Finally, we highlight how modulation of HO-1 activity regulates the mobilization of bone marrow hematopoietic cells to peripheral blood. We critically discuss the issue of metalloporphyrins, commonly used pharmacological modulators of HO-1 activity, and raise the issue of their important HO-1-independent activities.

Role of HMOX1 Promoter Genetic Variants in Chemoresistance and Chemotherapy Induced Neutropenia in Children with Acute Lymphoblastic Leukemia.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

Proximity Ligation Assay Detection of Protein-DNA Interactions-Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1-/- mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4-protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

Effect of Heme Oxygenase-1 on Melanoma Development in Mice-Role of Tumor-Infiltrating Immune Cells.

OBJECTIVE: Heme oxygenase-1 (HO-1) is a cytoprotective, proangiogenic and anti-inflammatory enzyme that is often upregulated in tumors. Overexpression of HO-1 in melanoma cells leads to enhanced tumor growth, augmented angiogenesis and resistance to anticancer treatment. The effect of HO-1 in host cells on tumor development is, however, hardly known. METHODS AND RESULTS: To clarify the effect of HO-1 expression in host cells on melanoma progression, C57BL/6xFvB mice of different HO-1 genotypes, HO-1+/+, HO-1+/-, and HO-1-/-, were injected with the syngeneic wild-type murine melanoma B16(F10) cell line. Lack of HO-1 in host cells did not significantly influence the host survival. Nevertheless, in comparison to the wild-type counterparts, the HO-1+/- and HO-1-/- males formed bigger tumors, and more numerous lung nodules; in addition, more of them had liver and spleen micrometastases. Females of all genotypes developed at least 10 times smaller tumors than males. Of importance, the growth of primary and secondary tumors was completely blocked in HO-1+/+ females. This was related to the increased infiltration of leukocytes (mainly lymphocytes T) in primary tumors. CONCLUSIONS: Although HO-1 overexpression in melanoma cells can enhance tumor progression in mice, its presence in host cells, including immune cells, can reduce growth and metastasis of melanoma.

Exacerbation of Neonatal Hemolysis and Impaired Renal Iron Handling in Heme Oxygenase 1-Deficient Mice.

In most mammals, neonatal intravascular hemolysis is a benign and moderate disorder that usually does not lead to anemia. During the neonatal period, kidneys play a key role in detoxification and recirculation of iron species released from red blood cells (RBC) and filtered out by glomeruli to the primary urine. Activity of heme oxygenase 1 (HO1), a heme-degrading enzyme localized in epithelial cells of proximal tubules, seems to be of critical importance for both processes. We show that, in HO1 knockout mouse newborns, hemolysis was prolonged despite a transient state and exacerbated, which led to temporal deterioration of RBC status. In neonates lacking HO1, functioning of renal molecular machinery responsible for iron reabsorption from the primary urine (megalin/cubilin complex) and its transfer to the blood (ferroportin) was either shifted in time or impaired, respectively. Those abnormalities resulted in iron loss from the body (excreted in urine) and in iron retention in the renal epithelium. We postulate that, as a consequence of these abnormalities, a tight systemic iron balance of HO1 knockout neonates may be temporarily affected.

HIF-1 stabilization exerts anticancer effects in breast cancer cells in vitro and in vivo.

Tumor hypoxia and high activity of hypoxia-inducible factor-1 (HIF-1) correlate with adverse disease outcomes, malignancy, resistance to therapy and metastasis. Nonetheless, recent studies indicate that under certain circumstances, HIF-1 stabilization may exert protective effects and even decrease tumor cell aggressiveness. This study aimed to characterize the potential anticancer effect of molidustat (BAY 85-3934), the prolyl hydroxylase (PHD) inhibitor and HIF-1 stabilizator. We confirmed that molidustat stabilizes HIF-1α and induces the expression of vascular endothelial growth factor (VEGF) in MDA-MB-231 breast cancer cells, to a similar or even greater extent than hypoxia. Interestingly, decreased cell survival and colony formation capabilities, together with S/G2 cell cycle arrest, were observed after treatment with PHD inhibitor. Importantly, molidustat enhanced the effectiveness of the chemotherapeutic drug, gemcitabine, on cancer cells. Finally, the xenograft model revealed decreased tumor growth in vivo after molidustat treatment. Both in vitro and in vivo analysis showed no differences in the angiogenic potential of endothelial cells treated with tumor-conditioned media or vascularization of the MDA-MB-231 xenografts, respectively. In summary, molidustat treatment exhibits an inhibitory effect on breast cancer cell survival, self-renewal capacity and potentiates the efficacy of chemotherapeutic gemcitabine.

Synthetically Lethal Interactions of Heme Oxygenase-1 and Fumarate Hydratase Genes.

Elevated expression of heme oxygenase-1 (HO-1, encoded by HMOX1) is observed in various types of tumors. Hence, it is suggested that HO-1 may serve as a potential target in anticancer therapies. A novel approach to inhibit HO-1 is related to the synthetic lethality of this enzyme and fumarate hydratase (FH). In the current study, we aimed to validate the effect of genetic and pharmacological inhibition of HO-1 in cells isolated from patients suffering from hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-an inherited cancer syndrome, caused by FH deficiency. Initially, we confirmed that UOK 262, UOK 268, and NCCFH1 cell lines are characterized by non-active FH enzyme, high expression of Nrf2 transcription factor-regulated genes, including HMOX1 and attenuated oxidative phosphorylation. Later, we demonstrated that shRNA-mediated genetic inhibition of HMOX1 resulted in diminished viability and proliferation of cancer cells. Chemical inhibition of HO activity using commercially available inhibitors, zinc and tin metalloporphyrins as well as recently described new imidazole-based compounds, especially SLV-11199, led to decreased cancer cell viability and clonogenic potential. In conclusion, the current study points out the possible relevance of HO-1 inhibition as a potential anti-cancer treatment in HLRCC. However, further studies revealing the molecular mechanisms are still needed.

miR-378a influences vascularization in skeletal muscles.

AIMS: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. METHODS AND RESULTS: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a-/-) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a-/- aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a-/- muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a-/- and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G-) was higher in the ischaemic muscles of miR-378a-/- mice, suggesting an anti-inflammatory action of miR-378a. CONCLUSIONS: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.

Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance.

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.

Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Atorvastatin and Conditioned Media from Atorvastatin-Treated Human Hematopoietic Stem/Progenitor-Derived Cells Show Proangiogenic Activity In Vitro but Not In Vivo.

Myeloid angiogenic cells (MAC) derive from hematopoietic stem/progenitor cells (HSPCs) that are mobilized from the bone marrow. They home to sites of neovascularization and contribute to angiogenesis by production of paracrine factors. The number and function of proangiogenic cells are impaired in patients with diabetes or cardiovascular diseases. Both conditions can be accompanied by decreased levels of heme oxygenase-1 (HMOX1), cytoprotective, heme-degrading enzyme. Our study is aimed at investigating whether precursors of myeloid angiogenic cells (PACs) treated with known pharmaceuticals would produce media with better proangiogenic activity in vitro and if such media can be used to stimulate blood vessel growth in vivo. We used G-CSF-mobilized CD34+ HSPCs, FACS-sorted from healthy donor peripheral blood mononuclear cells (PBMCs). Sorted cells were predominantly CD133+. CD34+ cells after six days in culture were stimulated with atorvastatin (AT), acetylsalicylic acid (ASA), sulforaphane (SR), resveratrol (RV), or metformin (Met) for 48 h. Conditioned media from such cells were then used to stimulate human aortic endothelial cells (HAoECs) to enhance tube-like structure formation in a Matrigel assay. The only stimulant that enhanced PAC paracrine angiogenic activity was atorvastatin, which also had ability to stabilize endothelial tubes in vitro. On the other hand, the only one that induced heme oxygenase-1 expression was sulforaphane, a known activator of a HMOX1 inducer-NRF2. None of the stimulants changed significantly the levels of 30 cytokines and growth factors tested with the multiplex test. Then, we used atorvastatin-stimulated cells or conditioned media from them in the Matrigel plug in vivo angiogenic assay. Neither AT alone in control media nor conditioned media nor AT-stimulated cells affected numbers of endothelial cells in the plug or plug's vascularization. Concluding, high concentrations of atorvastatin stabilize tubes and enhance the paracrine angiogenic activity of human PAC cells in vitro. However, the effect was not observed in vivo. Therefore, the use of conditioned media from atorvastatin-treated PAC is not a promising therapeutic strategy to enhance angiogenesis.

Role of the kidneys in the redistribution of heme-derived iron during neonatal hemolysis in mice.

Moderate intravascular hemolysis is a common condition in newborns. It is followed by the accumulation of bilirubin, which is a secondary product of the activity of heme oxygenase-1, an enzyme that catalyzes the breakdown of heme released from disrupted erythrocytes and taken up by hepatic macrophages. Although these cells are a major site of enzymatic heme breakdown in adults, we show here that epithelial cells of proximal tubules in the kidneys perform the functions of both heme uptake and catabolism in mouse neonates. A time-course study examining mouse pups during the neonatal period showed a gradual recovery from hemolysis, and concomitant decreases in the expression of heme-related genes and non-heme iron transporters in the proximal tubules. By adjusting the expression of iron-handling proteins in response to the disappearance of hemolysis in mouse neonates, the kidneys may play a role in the detoxification of iron and contribute to its recirculation from the primary urine to the blood.