Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

Background Endothelial progenitor cell (EPC) dysfunction contributes to vascular disease in diabetes mellitus. However, the molecular mechanism underlying EPC dysfunction and its contribution to delayed reendothelialization in diabetes mellitus remain unclear. Our study aimed to illustrate the potential molecular mechanism underlying diabetic EPC dysfunction in vivo and in vitro. Furthermore, we assessed the effect of EPC transplantation on endothelial regeneration in diabetic rats. Methods Late outgrowth EPCs were isolated from the bone marrow of rats for in vivo and in vitro studies. In vitro functional assays and Western blotting were conducted to reveal the association between C-X-C chemokine receptor type 7 (CXCR7) expression and diabetic EPC dysfunction. To confirm the association between cellular CXCR7 levels and EPC function, CXCR7 expression in EPCs was upregulated and downregulated via lentiviral transduction and RNA interference, respectively. Western blotting was used to reveal the potential molecular mechanism by which the Stromal-Derived Factor-1 (SDF-1)/CXCR7 axis regulates EPC function. To elucidate the role of the SDF-1/CXCR7 axis in EPC-mediated endothelial regeneration, a carotid artery injury model was established in diabetic rats. After the model was established, saline-treated, diabetic, normal, or CXCR7-primed EPCs were injected via the tail vein. Results Diabetic EPC dysfunction was associated with decreased CXCR7 expression. Furthermore, EPC dysfunction was mimicked by knockdown of CXCR7 in normal EPCs. However, upregulating CXCR7 expression reversed the dysfunction of diabetic EPCs. The SDF-1/CXCR7 axis positively regulated EPC function by activating the AKT-associated Kelch-like ECH-associated protein 1 (keap-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis, which was reversed by blockade of AKT and Nrf2. Transplantation of CXCR7-EPCs accelerated endothelial repair and attenuated neointimal hyperplasia in diabetes mellitus more significantly than transplantation of diabetic or normal EPCs. However, the therapeutic effect of CXCR7-EPC transplantation on endothelial regeneration was reversed by knockdown of Nrf2 expression. Conclusions Dysfunction of diabetic EPCs is associated with decreased CXCR7 expression. Furthermore, the SDF-1/CXCR7 axis positively regulates EPC function by activating the AKT/keap-1/Nrf2 axis. CXCR7-primed EPCs might be useful for endothelial regeneration in diabetes-associated vascular disease.

Decitabine Reactivation of FoxM1-Dependent Endothelial Regeneration and Vascular Repair for Potential Treatment of Elderly ARDS and COVID-19 Patients

Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS) and COVID-19. We repot that aging impairs the intrinsic FoxM1-dependent endothelial regeneration and vascular repair program and causes persistent lung injury and high mortality following sepsis. Therapeutic gene transduction of FOXM1 in vascular endothelium or treatment with FDA-approved drug Decitabine was sufficient to reactivate FoxM1-dependent lung endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular endothelial cells of elderly patients in contrast to mid-age patients. Thus, Decitabine reactivation of FoxM1-dependent vascular repair represents a potential effective therapy for elderly COVID-19 and non-COVID-19 ARDS patients.

A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

Background Failure of the corneal endothelium cell (CEC) monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form CEC monolayer. We hypothesize that by using dental pulp stem cells (DPSC) that share an embryological origin with CEC, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells (iPSC). Methods In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem cells and, then, into CEC. Results Initially, we compared the efficiency of direct differentiation of DPSC with the differentiation of iPSC to express NCSC related genes in adhesion culture, showing significantly higher levels of early stage marker AP2 for the DPSC compared to iPSC. To provide better environment for NCSC gene expression, suspension method was performed, which induced the formation of neurospheres. The results showed neurosphere formation after few days, obtaining the peak of NCSC marker expression after 4 days, showing overexpression of AP2, p75 and CHD7 markers, confirming the formation of NCSC like cells. Furthermore, pluripotent markers Oct4, Nanog and Sox2 were as well upregulated in suspension culture. Neurospheres were then directly cultured in CEC conditioned medium for the second differentiation, showing the conversion of DPSC into polygonal like cells expressing higher levels of ZO-1, ATP1A1, COL4A2 and COL8A1 markers, providing proof of the successful conversion into CEC. Conclusions Therefore, our findings demonstrate that patient-derived dental pulp stem cells represent an autologous cell source for corneal endothelial regeneration that avoids actual transplantation limitations as well as reprogramming techniques.

Decreased Jagged1 expression in vascular smooth muscle cells delays endothelial regeneration in arteriovenous graft

Aims It is well-established that endothelial dysfunction promotes activation of vascular smooth muscle cell (VSMC). Whether decreased accumulation of VSMCs affects endothelial regeneration and functions in arteriovenous graft (AVG) remodelling has not been studied. We sought to identify mechanisms by which the Notch ligand, Jagged1, in VSMCs regulates endothelial cell (EC) functions in AVGs. Methods and results AVGs were created in transgenic mice bearing VSMC-specific knockout (KO) or overexpression of Jagged1. VSMC migration, EC regeneration, and its barrier functions as well as AVG remodelling were evaluated. Jagged1 expression was induced in VSMCs of neointima in the AVGs. Jagged1 KO in VSMCs inhibited the accumulation of extracellular matrix as well as VSMC migration. Fewer α-SMA-positive VSMCs were found in AVGs created in VSMC-specific Jagged1 KO mice (VSMCJagged1 KO mice) vs. in WT mice. Decreased VSMCs in AVGs were associated with deterioration of EC functions. In AVGs created in transgenic mice bearing Jagged1 KO in VSMCs exhibited delayed EC regeneration and impaired EC barrier function. Barrier dysfunction of ECs increased inflammatory cell infiltration and dysregulation of AVG remodelling and arterialization. The increased expression of IL-1β in macrophages was associated with expression of adhesion markers in ECs in AVGs created in VSMCJagged1 KO mice. In contrast, AVGs created in mice with overexpression of Jagged1 in VSMCs exhibited improved EC regeneration plus decreased macrophage infiltration. This led to AVG remodelling and arterialization. In co-cultures of ECs and VSMCs, Jagged1 deficiency in VSMCs suppressed N-cadherin and integrin β3 expression in ECs. Inhibition of integrin β3 activation delayed EC spreading and migration. Notably, Jagged1 overexpression in VSMCs or treatment with recombinant Jagged1 stimulated the expression of N-cadherin and integrin β3 in ECs. Jagged1-induced responses were blocked by inhibition of Notch signalling. Conclusions Jagged1 expression in VSMCs maintains EC barrier functions and blocks infiltration of macrophages. These responses promote remodelling and arterialization of AVGs.