Micro- and Macrovascular Effects of Inflammation in Peripheral Artery Disease-Pathophysiology and Translational Therapeutic Approaches.

Inflammation has a critical role in the development and progression of atherosclerosis. On the molecular level, inflammatory pathways negatively impact endothelial barrier properties and thus, tissue homeostasis. Conformational changes and destruction of the glycocalyx further promote pro-inflammatory pathways also contributing to pro-coagulability and a prothrombotic state. In addition, changes in the extracellular matrix composition lead to (peri-)vascular remodelling and alterations of the vessel wall, e.g., aneurysm formation. Moreover, progressive fibrosis leads to reduced tissue perfusion due to loss of functional capillaries. The present review aims at discussing the molecular and clinical effects of inflammatory processes on the micro- and macrovasculature with a focus on peripheral artery disease.

Toll-like Receptors as Pro-Thrombotic Drivers in Viral Infections: A Narrative Review.

Toll-like receptors (TLRs) have a critical role in the pathogenesis and disease course of viral infections. The induced pro-inflammatory responses result in the disturbance of the endovascular surface layer and impair vascular homeostasis. The injury of the vessel wall further promotes pro-thrombotic and pro-coagulatory processes, eventually leading to micro-vessel plugging and tissue necrosis. Moreover, TLRs have a direct role in the sensing of viruses and platelet activation. TLR-mediated upregulation of von Willebrand factor release and neutrophil, as well as macrophage extra-cellular trap formation, further contribute to (micro-) thrombotic processes during inflammation. The following review focuses on TLR signaling pathways of TLRs expressed in humans provoking pro-thrombotic responses, which determine patient outcome during viral infections, especially in those with cardiovascular diseases.

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.

Microvascular Thrombosis as a Critical Factor in Severe COVID-19.

Platelet-endothelial interactions have a critical role in microcirculatory function, which maintains tissue homeostasis. The subtle equilibrium between platelets and the vessel wall is disturbed by the coronavirus disease 2019 (COVID-19), which affects all three components of Virchow's triad (endothelial injury, stasis and a hypercoagulable state). Endotheliitis, vasculitis, glycocalyx degradation, alterations in blood flow and viscosity, neutrophil extracellular trap formation and microparticle shedding are only few pathomechanisms contributing to endothelial damage and microthrombosis resulting in capillary plugging and tissue ischemia. In the following opinion paper, we discuss major pathological processes leading to microvascular endothelial activation and thrombosis formation as a possible major adverse factor driving the deterioration of patient disease course in severe COVID-19.

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.

NRF2 Regulates Viability, Proliferation, Resistance to Oxidative Stress, and Differentiation of Murine Myoblasts and Muscle Satellite Cells.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

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.

Nrf2 Transcriptional Activity Governs Intestine Development.

Our recent findings indicate that Nrf2 transcriptional activity is essential in maintaining the proper large intestinal structure in adult mice. Here, we aimed to verify whether Nrf2-related intestine abnormalities stemmed from the early weaning or gestational periods. Therefore, we analyzed 4-day-old pups and embryos devoid of Nrf2 transcriptional activity (tKO) and their wild-type counterparts. We found significant changes in the intestinal structure of 4-day-old Nrf2 tKO pups including a longer colon, altered crypt distribution, and enlargement of the goblet cells with a markedly higher level of mucin 2. Tracing back the origin of these alterations, we observed that they appeared as early as day 14.5 of embryonic development, independently of sex. Importantly, in this period, we observed a significant increase in the Nrf2 level and a distinctive, untimely pattern of expression of the proliferation factor Ki67. At the latest stage of embryonic development, we detected a premature drop in the differentiation factor Notch1. We suspect that intestine abnormalities in mice lacking Nrf2 transcriptional activity stem from sex-independent disturbed intestinal cell proliferation and could be further exacerbated by altered differentiation. Summing up, we identified Nrf2 transcriptional activity as an important regulator of intestinal formation. It influences the hindgut cell proliferation and differentiation at different stages of embryonic development.

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.

miR-378 affects metabolic disturbances in the mdx model of Duchenne muscular dystrophy.

Although Duchenne muscular dystrophy (DMD) primarily affects muscle tissues, the alterations to systemic metabolism manifested in DMD patients contribute to the severe phenotype of this fatal disorder. We propose that microRNA-378a (miR-378) alters carbohydrate and lipid metabolism in dystrophic mdx mice. In our study, we utilized double knockout animals which lacked both dystrophin and miR-378 (mdx/miR-378-/-). RNA sequencing of the liver identified 561 and 194 differentially expressed genes that distinguished mdx versus wild-type (WT) and mdx/miR-378-/- versus mdx counterparts, respectively. Bioinformatics analysis predicted, among others, carbohydrate metabolism disorder in dystrophic mice, as functionally proven by impaired glucose tolerance and insulin sensitivity. The lack of miR-378 in mdx animals mitigated those effects with a faster glucose clearance in a glucose tolerance test (GTT) and normalization of liver glycogen levels. The absence of miR-378 also restored the expression of genes regulating lipid homeostasis, such as Acly, Fasn, Gpam, Pnpla3, and Scd1. In conclusion, we report for the first time that miR-378 loss results in increased systemic metabolism of mdx mice. Together with our previous finding, demonstrating alleviation of the muscle-related symptoms of DMD, we propose that the inhibition of miR-378 may represent a new strategy to attenuate the multifaceted symptoms of DMD.

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.

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.

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.

A Dual Role of Heme Oxygenase-1 in Angiotensin II-Induced Abdominal Aortic Aneurysm in the Normolipidemic Mice.

Abdominal aortic aneurysm (AAA) bears a high risk of rupture and sudden death of the patient. The pathogenic mechanisms of AAA remain elusive, and surgical intervention represents the only treatment option. Heme oxygenase-1 (HO-1), a heme degrading enzyme, is induced in AAA, both in mice and humans. HO-1 was reported to mitigate AAA development in an angiotensin II (AngII)-induced model of AAA in hyperlipidemic ApoE-/- mice. Since the role of hyperlipidaemia in the pathogenesis of AAA remains controversial, we aimed to evaluate the significance of HO-1 in the development and progression of AAA in normolipidemic animals. The experiments were performed in HO-1-deficient mice and their wild-type counterparts. We demonstrated in non-hypercholesterolemic mice that the high-dose of AngII leads to the efficient formation of AAA, which is attenuated by HO-1 deficiency. Yet, if formed, they are significantly more prone to rupture upon HO-1 shortage. Differential susceptibility to AAA formation does not rely on enhanced inflammatory response or oxidative stress. AAA-resistant mice are characterized by an increase in regulators of aortic remodeling and angiotensin receptor-2 expression, significant medial thickening, and delayed blood pressure elevation in response to AngII. To conclude, we unveil a dual role of HO-1 deficiency in AAA in normolipidemic mice, where it protects against AAA development, but exacerbates the state of formed AAA.

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.