Publications

Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.

<p>Comparison of SMS-CTR eRMS (SMS) and CW9019 aRMS (CW) cell lines cultured under standard conditions. Expression of VEGF, VEGFR-1, VEGFR-2, HIF-1, HIF-2 and miR-146a genes.</p>

Wound healing is an intricate process requiring the concerted action of keratinocytes, fibroblasts, endothelial cells, and macrophages. Here, we review the literature on normal wound healing and the pathological forms of wound healing, such as hypertrophic or excessive scar formation, with special emphasis on the heme-heme oxygenase (HO) system and the versatile effector molecules that are formed after HO-mediated heme degradation. Excessive scar formation following wounding is thought to relate to prolonged oxidative and inflammatory stress in the skin. Evidence is accumulating that the heme-HO system forms a novel and important target in the control of wound healing. Heme-protein derived heme can act as a potent oxidative and inflammatory stress inducer, and excess levels of heme may thus contribute to delayed resolution of oxidative and inflammatory insults in the skin. This emphasizes the need for a timely reduction of the levels of heme. Heme-binding proteins, heme transporters, and the heme degrading protein, HO, form therefore a necessary defense. Deficiencies in these defense proteins or a disturbed redox status, as in diabetic patients, may render individuals more prone to heme-induced deleterious effects. A better understanding of the heme-heme oxygenase system as target during wound healing may result in novel strategies to reduce scar formation.

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.

Cellular retinoic acid binding proteins are considered to be involved in retinoic acid (RA) signaling pathways. Our aim was to compare the expression and localization of cellular retinoic acid binding proteins I and II (CRABP I and II) in embryonic mouse hearts during normal development and after a single teratogenic dose of RA. Techniques such as real-time PCR, RT-PCR, Western blots and immunostaining were employed to examine hearts from embryos at 9-17 dpc. RA treatment at 8.5dpc affects production of CRABP I and II in the heart in the 48-h period. Changes in expression of mRNA for retinaldehyde dehydrogenase II (Raldh2), Crabp1 and Crabp2 genes also occur within the same time window (i.e. 10-11dpc) after RA treatment. In the embryonic control heart these proteins are localized in groups of cells within the outflow tract (OT), and the atrioventricular endocardial cushions. A gradient of labeling is observed with CRABP II but not for CRABP I along the myocardium of the looped heart at 11 dpc; this gradient is abolished in hearts treated with RA, whereas an increase of RALDH2 staining has been observed at 10 dpc in RA-treated hearts. Some populations of endocardial endothelial cells were intensively stained with anti-CRABP II whereas CRABP I was negative in these structures. These results suggest that CRABP I and II are independently regulated during heart development, playing different roles in RA signaling, essential for early remodeling of the heart tube and alignment of the great arteries to their respective ventricles.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Young scientists grant MNS9/2021 Polish National Science Centre PRELUDIUM grant 2019/33/N/NZ1/03066 Introduction Although gene therapy has become a feasible alternative to regular treatment in numerous disorders, efficient targeting of human heart still seems out of reach. So far, adeno-associated viral vectors (AAVs) have been the most promising transgene carriers in in vivo applications. Transduction with AAVs relies on serotype-specific binding to the glycan residues on the cell surface. While AAV serotype 9 (AAV9) - binding galactose - was successfully used for delivery of therapeutic genes to cardiac muscle in murine models, its effect was underwhelming in large animals. Switching the serotype to AAV1 or AAV6 – binding sialic acids – enabled transduction of porcine hearts, and cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) in 2D in vitro culture. But, as evidenced in clinical trials where less than 1% of heart cells contained delivered transgene, such strategy was not suitable for in vivo application in humans. Purpose Taking into account differences in glycosylation patterns between humans and other mammals, we aimed to investigate the role of surface glycans in AAV6 and AAV9 transduction mechanism in 2D and 3D cultures of human cardiomyocytes. Methods We generated hiPSC- derived cardiomyocytes (hiPSC-CMs) and epicardial fibroblasts (hiPSC-CFs). Cells were cultured in regular 2D conditions or 3D co-cultures (as spheroids), since direct cells interaction may influence the availability of terminal sugar residues. Surface glycans were stained with lectins and imaged using confocal microscope. Results While in 2D conditions AAV6 transduces hiPSC-CMs very efficiently, it is significantly less potent in 3D culture. Conversely, AAV9 maintains most of its functionality in 3D model. Our data collected from spheroids, indicate that cells in such 3D cultures undergo significant changes in terminal glycosylation pattern. While the level of galactose (AAV9 receptor) increased from day 1 to day 7, the signal from sialic acids attached to galactose (AAV6 receptor) decreased. This effect was accompanied by the upregulation of sialidase-3 expression, that regulates availability of galactose residues on the cell surface. As a result, transduction efficiency was improved in spheroids exposed to AAV9 on day 7 of 3D culture, in comparison to those transduced on day 1. Inhibition of sialidases activity with NADNA reduced the level of terminal galactose in both, hiPSC-CMs and hiPSC-CFs, and was associated with more efficient transduction of cardiomyocytes with AAV6, and less efficient transduction with AAV9. Additionally, we observed that galactosylation of collagen can be another factor that influences cell-vector interaction in 2D and 3D culture. Conclusions Our results demonstrate that the availability of terminal sugar residues is a key factor regulating transduction of hiPSC-CMs with AAV vectors, and underline the need for development of appropriate models for testing of AAV functionality in preclinical studies.