Publications

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.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre Maturity Onset Diabetes of the Young (MODY) is an autosomal inherited disease and its most prevalent form is caused by mutations in the hepatocyte nuclear factor 1-alpha (HNF1A). HNF1A-MODY patients often develop vascular complications and have increased incidents of cardiovascular-related death. In our recent work (1), we have established a disease model based on human induced pluripotent stem cells (hiPSCs) derived from a healthy donor and isogenic lines with the mutations either in one of the HNF1A alleles (monoallelic, MAC) or in both of them (biallelic, BAC). After differentiation of these lines toward endothelial cells (hiPSC-ECs), we have shown that HNF1A-mutated hiPSC-ECs have increased permeability after TNFα stimulation. Interleukin 8 (IL-8) could be an important player in this process (2), but its possible role in HNF1A-related increased permeability has not been studied yet. The aim of this study was to evaluate whether increased vascular permeability in HNF1A-mutated hiPSC-ECs could be related to IL-8 production of these cells. Results were based on our previously described isogenic hiPSC-ECs. IL-8 production was assessed with ELISA, proliferation through BrdU incorporation test and apoptosis was evaluated with PO-PRO1/7-AAD kit. There was an increased IL-8 production in BAC line under basic conditions, meaning that these cells present more activated proinflammatory status. After stimulation with TNFα, all hiPSC-ECs increased IL-8 levels, however HNF1A-mutated cells were less efficient. BAC hiPSC-ECs had the lowest IL-8 production after TNFα, meaning that increased vascular permeability in these cells could not be related to IL-8. As IL-8 has also an important role in the proliferation and survival of the ECs (3), we checked whether increased IL-8 production under basic conditions in BAC could have an impact on the proliferation and apoptosis of these cells. The BAC hiPSC-ECs had significantly decreased proliferation compared to both control and MAC lines. Additionally, both HNF1A-mutated lines had an increase in the percentage of the apoptotic cells. After stimulation with TNFα, BAC hiPSC-ECs failed to further increase the percentage of the apoptotic cells, as compared to MAC and control cells. Summarizing, the increased vascular permeability of HNF1A-mutated hiPSC-ECs in response to TNFα could not be attributed to IL-8. The increased IL-8 of BAC hiPSC-ECs under basic conditions did not improve their proliferation or survival capacity. Contrary, these cells showed decreased proliferation and increased percentage of apoptotic cells. Moreover, in response to proinflammatory cytokine, BAC hiPSC-ECs failed to further increase the percentage of the apoptotic cells, which could be related to alterations in the cell cycle of these cells. All these results show that HNF1A-mutated iPSC-ECs have impaired response to TNFα, which may contribute to increased incidence of vascular complications related to HNF1A-MODY.

Nrf2 transcription factor plays a crucial role in protection of cells against oxidative stress. Under normal conditions Nrf2 is sequestered in cytoplasm by a cytoskeletal protein, Keapl. Situation is changed under stressful conditions,when electrophiles and/or reactiveoxygen species (ROS) cause dissociation of Nrf2 from Keap1. As a consequence, Nrf2 is translocated to the nucleus, that leads to activation of cytoprotective genes involved in electrophile conjugation, excretion of xenobiotics, ROS scavenging and stabilization of cellular redox potential. Amongst xenobiotics, there are many mutagenic and cancerogenic factors. Phase I and II enzymes are responsible for inactivation and removal of such compounds. It has been shown that induction of phase II enzymes confers protection upon insult by reactive metabolites of cancerogens or ROS. Since Nrf2 is an inductor of those enzymes it can be considered as a potential target for cancer chemoprevention. Similarly, in case of neurodegenerative disorders, which pathogenesis is connected to oxidative stress, Nrf2 may be important for therapeutical and preventive strategies

Abstract Aims/Introduction Type 2 diabetes is often complicated by diabetic foot syndrome ( DFS ). We analyzed the circulating stem cells, growth factor and anti‐oxidant gene expression profiles in type 2 diabetes patients without or with different forms of DFS . Materials and Methods Healthy volunteers ( n = 13) and type 2 diabetes patients: (i) without DFS ( n = 10); or with (ii) Charcot osteoneuropathy ( n = 10); (iii) non‐infected ( n = 17); (iv) infected ( n = 11); and (v) healed ulceration were examined ( n = 12). Peripheral blood endothelial progenitor cells ( EPC ), mesenchymal stem cells ( MSC ), hematopoietic stem cells ( HSC ) and very small embryonic‐like ( VSEL ) cells were phenotyped using flow cytometry. Plasma cytokine concentrations and gene expressions in blood cells were measured by L uminex and quantitative real‐time polymerase chain reaction assays, respectively. Results Patients with non‐complicated type 2 diabetes showed reduced HMOX 1 expression, accompanied by HMOX 2 upregulation, and had less circulating EPC , MSC or HSC than healthy subjects. In contrast, VSEL cells were elevated in the type 2 diabetes group. However, subjects with DFS , even with healed ulceration, had fewer VSEL cells, more CD 45‐ CD 29 + CD 90 + MSC , and upregulated HMOX 1 when compared with the type 2 diabetes group. Patients with C harcot osteopathy had lowered plasma fibroblast growth factor‐2. Elevated plasma tumor necrosis factor‐α and decreased catalase expression was found in all diabetic patients. Conclusions Patients with type 2 diabetes and different forms of DFS have an altered number of circulating stem cells. Type 2 diabetes might also be associated with a changed plasma growth factor and anti‐oxidant gene expression profile. Altogether, these factors could contribute to the pathogenesis of different forms of DFS .