Casein kinase 2 activity is a host restriction factor for AAV transduction.

So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.

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.

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.

Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors.

Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.

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.

Simvastatin Attenuates Abdominal Aortic Aneurysm Formation Favoured by Lack of Nrf2 Transcriptional Activity.

Surgical intervention is currently the only option for an abdominal aortic aneurysm (AAA), preventing its rupture and sudden death of a patient. Therefore, it is crucial to determine the pathogenic mechanisms of this disease for the development of effective pharmacological therapies. Oxidative stress is said to be one of the pivotal factors in the pathogenesis of AAAs. Thus, we aimed to evaluate the significance of nuclear factor erythroid 2-related factor 2 (Nrf2) transcriptional activity in the development of AAA and to verify if simvastatin, administered as pre- and cotreatment, may counteract this structural malformation. Experiments were performed on mice with inhibited transcriptional activity of Nrf2 (tKO) and wild-type (WT) counterparts. We used a model of angiotensin II- (AngII-) induced AAA, combined with a fat-enriched diet. Mice were administered with AngII or saline for up to 28 days via osmotic minipumps. Simvastatin administration was started 7 days before the osmotic pump placement and then continued until the end of the experiment. We found that Nrf2 inactivation increased the risk of development and rupture of AAA. Importantly, these effects were reversed by simvastatin in tKO mice, but not in WT. The abrupt blood pressure rise induced by AngII was mitigated in simvastatin-treated animals regardless of the genotype. Simvastatin-affected parameters that differed between the healthy structure of the aorta and aneurysmal tissue included immune cell infiltration of the aortic wall, VCAM1 mRNA and protein level, extracellular matrix degradation, TGF-ß1 mRNA level, and ERK phosphorylation, but neither oxidative stress nor the level of Angiotensin II Type 1 Receptor (AT1R). Taken together, the inhibition of Nrf2 transcriptional activity facilitates AAA formation in mice, which can be prevented by simvastatin. It suggests that statin treatment of patients with hypercholesterolemia might have not only a beneficial effect in terms of controlling atherosclerosis but also potential AAA prevention.

Keap1 governs ageing-induced protein aggregation in endothelial cells.

The breach of proteostasis, leading to the accumulation of protein aggregates, is a hallmark of ageing and age-associated disorders, up to now well-established in neurodegeneration. Few studies have addressed the issue of dysfunctional cell response to protein deposition also for the cardiovascular system. However, the molecular basis of proteostasis decline in vascular cells, as well as its relation to ageing, are not understood. Recent studies have indicated the associations of Nrf2 transcription factor, the critical modulator of cellular stress-response, with ageing and premature senescence. In this report, we outline the significance of protein aggregation in physiological and premature ageing of murine and human endothelial cells (ECs). Our study shows that aged donor-derived and prematurely senescent Nrf2-deficient primary human ECs, but not those overexpressing dominant-negative Nrf2, exhibit increased accumulation of protein aggregates. Such phenotype is also found in the aortas of aged mice and young Nrf2 tKO mice. Ageing-related loss of proteostasis in ECs depends on Keap1, well-known repressor of Nrf2, recently perceived as a key independent regulator of EC function and protein S-nitrosation (SNO). Deposition of protein aggregates in ECs is associated with impaired autophagy. It can be counteracted by Keap1 depletion, S-nitrosothiol reductant or rapamycin treatment. Our results show that Keap1:Nrf2 protein balance and Keap1-dependent SNO predominate Nrf2 transcriptional activity-driven mechanisms in governing proteostasis in ageing ECs.

Beyond repression of Nrf2: An update on Keap1.

Nrf2 (NFE2L2 - nuclear factor (erythroid-derived 2)-like 2) is a transcription factor, which is repressed by interaction with a redox-sensitive protein Keap1 (Kelch-like ECH-associated protein 1). Deregulation of Nrf2 transcriptional activity has been described in the pathogenesis of multiple diseases, and the Nrf2/Keap1 axis has emerged as a crucial modulator of cellular homeostasis. Whereas the significance of Nrf2 in the modulation of biological processes has been well established and broadly discussed in detail, the focus on Keap1 rarely goes beyond the regulation of Nrf2 activity and redox sensing. However, recent studies and scrutinized analysis of available data point to Keap1 as an intriguing and potent regulator of cellular function. This review aims to shed more light on Keap1 structure, interactome, regulation and non-canonical functions, thereby enhancing its significance in cell biology. We also intend to highlight the impact of balance between Keap1 and Nrf2 in the maintenance of cellular homeostasis.

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.

Keap1 controls protein S-nitrosation and apoptosis-senescence switch in endothelial cells.

Premature senescence, a death escaping pathway for cells experiencing stress, is conducive to aging and cardiovascular diseases. The molecular switch between senescent and apoptotic fate remains, however, poorly recognized. Nrf2 is an important transcription factor orchestrating adaptive response to cellular stress. Here, we show that both human primary endothelial cells (ECs) and murine aortas lacking Nrf2 signaling are senescent but unexpectedly do not encounter damaging oxidative stress. Instead, they exhibit markedly increased S-nitrosation of proteins. A functional role of S-nitrosation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage and redirection of ECs to premature senescence. S-nitrosation and senescence are mediated by Keap1, a direct binding partner of Nrf2, which colocalizes and precipitates with nitric oxide synthase (NOS) and transnitrosating protein GAPDH in ECs devoid of Nrf2. We conclude that the overabundance of this "unrestrained" Keap1 determines the fate of ECs by regulation of S-nitrosation and propose that Keap1/GAPDH/NOS complex may serve as an enzymatic machinery for S-nitrosation in mammalian cells.

Biliverdin reductase deficiency triggers an endothelial-to-mesenchymal transition in human endothelial cells.

Endothelial dysfunction accompanied by the loss of endothelial cell phenotype plays an essential role in cardiovascular diseases. Here, we report that knockdown of biliverdin reductase (BVR), the enzyme of the heme degradation pathway converting biliverdin to bilirubin, shifts endothelial phenotype of the primary human aortic endothelial cells (HAECs) to mesenchymal-like one. It is reflected by the loss of endothelial markers and angiogenic response, with concomitant acquiring of mesenchymal markers, increased migratory capacity and metalloproteinase activity. BVR-deficiency induces the activity of Nrf2 transcription factor and increases heme oxygenase-1 (HO-1) level, which is accompanied by the reduction of cellular heme content, increase in a free iron fraction and oxidative stress. Accordingly, the phenotype of BVR-deficient cells can be mimicked by hemin or iron overload. Depletion of HO-1 in BVR-deficient ECs abrogates the increase in intracellular free iron and oxidative stress, preventing the loss of endothelial markers. Treatment of BVR-deficient cells with bilirubin does not rescue the endothelial phenotype of HAECs. Unlike BLVRA mRNA level, the expression of HMOX1, HMOX1:BLVRA ratio and HO-1 protein level positively correlate with abdominal aortic aneurysm size in clinical samples. Collectively, the non-enzymatic activity of BVR contributes to the maintenance of healthy endothelial phenotype through the prevention of HO-1-dependent iron-overload, oxidative stress and subsequent endothelial-to-mesenchymal transition (EndMT).

Nrf2 Sequesters Keap1 Preventing Podosome Disassembly: A Quintessential Duet Moonlights in Endothelium.

AIMS: Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2 either stimulatory or repressive influence on angiogenesis. Our findings unveil the mechanism of Nrf2-dependent vessel formation, which reaches beyond transactivation of gene expression and reconciles previous discrepancies. RESULTS: We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system.

Nrf2 in aging - Focus on the cardiovascular system.

Aging is the most critical risk factor for the development of cardiovascular diseases and their complications. Therefore, the fine-tuning of cellular response to getting older is an essential target for prospective therapies in cardiovascular medicine. One of the most promising targets might be the transcription factor Nrf2, which drives the expression of cytoprotective and antioxidative genes. Importantly, Nrf2 expression correlates with potential lifespan in rodents. However, the effect of Nrf2 activity in vascular diseases might be ambiguous and strongly depend on the cell type. On the one hand, the Nrf2 activity may protect cells from oxidative stress and senescence, on the other hand, total lack of Nrf2 is protective against atherosclerosis development. Therefore, this review aims to discuss the current knowledge on the role played by the transcription factor Nrf2 in cardiovascular diseases and its potential effects on aging.

Simvastatin Treatment Upregulates HO-1 in Patients with Abdominal Aortic Aneurysm but Independently of Nrf2.

Heme oxygenase-1 (HO-1), encoded by HMOX1 gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin (N = 28) or without statins (N = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes HMOX1, NQO1, and GCLM expression remained unchanged in AAA. In vitro studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although HMOX1 expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs in vitro is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including HMOX1, in AAA tissue denotes Nrf2 independency.

Murine Bone Marrow Mesenchymal Stromal Cells Respond Efficiently to Oxidative Stress Despite the Low Level of Heme Oxygenases 1 and 2.

AIMS: Mesenchymal stromal cells (MSCs) are heterogeneous cells from adult tissues that are able to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes. Such cells are widely studied in regenerative medicine. However, the success of cellular therapy depends on the cell survival. Heme oxygenase-1 (HO-1, encoded by the Hmox1 gene), an enzyme converting heme to biliverdin, carbon monoxide, and Fe2+, is cytoprotective and can affect stem cell performance. Therefore, our study aimed at assessing whether Hmox1 is critical for survival and functions of murine bone marrow MSCs. RESULTS: Both MSC Hmox1+/+ and Hmox1-/- showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1+/+ and Hmox1-/- cells showed similar survival in response to 50 µmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1-/- bone marrow-derived proangiogenic cells (BDMC). Hmox1+/+ MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 µmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1-/- treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1-/- do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1-/- show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.