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Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class Ila HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class Ila HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease., Introduction Endothelial-mesenchymal transition (EndMT) is an essential process during cardiac development, contributing to cardiac valve formation and the stabilization of new vasculature in the developing embryo (1, 2). EndMT also [...]

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, reduces lower limb amputations in patients with type 2 diabetes. The mechanism is, however, unknown. In this study, we demonstrate that fenofibrate markedly attenuates diabetes-related impairment of ischemia-mediated angiogenesis. In a murine model of hindlimb ischemia, daily oral fenofibrate treatment restored diabetes-impaired blood flow recovery, foot movement, hindlimb capillary density, vessel diameter, and vascular endothelial growth factor signaling to nondiabetic levels in both wild-type and PPARα-knockout mice, indicating that these fenofibrate effects are largely PPARα independent. In vitro, fenofibric acid (FFA) rescued high glucose-induced (25 mmol/L) impairment of endothelial cell migration, tubulogenesis, and survival in a PPARα-independent manner. Interestingly, fenofibrate in vivo and FFA in vitro reversed high glucose-induced expression of thioredoxin-interacting protein (TXNIP), an exquisitely glucose-inducible gene previously identified as a critical mediator of diabetes-related impairment in neovascularization. Conversely, adenoviral overexpression of TXNIP abrogated the restorative effects of FFA on high glucose-impaired endothelial cell function in vitro, indicating that the effects of FFA are mediated by TXNIP. We conclude that fenofibrate rescues diabetic impairment in ischemia-mediated angiogenesis, in large part, by PPARα-independent regulation of TXNIP. These findings may therefore explain the reduction in amputations seen in patients with diabetes treated with fenofibrate. [ABSTRACT FROM AUTHOR]

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose-induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI(-/-) diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications. [ABSTRACT FROM AUTHOR]

The glycocalyx of the uterine luminal epithelium in the rat undergoes considerable reduction before implantation. In particular, the reduction of some mucins is necessary to facilitate blastocyst adhesion and subsequent implantation. The present study investigated the localisation, abundance and hormonal control of two mucin proteins, Muc13 and Muc15, in rat uterine epithelial cells during early pregnancy to determine whether they are likely to play a role in uterine receptivity for implantation. Muc13 and Muc15 are localised to the uterine luminal epithelium but show a presence and an absence, respectively, at the apical cell surface at the time of implantation. This localisation corresponds to changes in the molecular weights of Muc13 and Muc15, as shown with western blotting analysis. Furthermore, the localisation of Muc13 and Muc15 was shown to be controlled by the ovarian hormones, oestrogen and progesterone, and they were also localised in preimplantation rat blastocysts. Our results suggest that Muc15 may operate in an anti-adhesive capacity to prevent implantation while Muc13 potentially functions in either an adhesive or cell-signalling role in the events of implantation. At the time of implantation the uterus allows blastocyst implantation; however, at other times of the cycle, it prevents implantation. Mucins are a family of cell-surface proteins that normally impede successful implantation. This study found that mucin 13 may support implantation while mucin 15 may prevent implantation. These new findings in the rat uterus add to the knowledge of factors that promote implantation as well as prevent implantation, which could be useful targets for reproductive technology. [ABSTRACT FROM AUTHOR]

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, reduces lower limb amputations in patients with type 2 diabetes. The mechanism is, however, unknown. In this study, we demonstrate that fenofibrate markedly attenuates diabetes-related impairment of ischemia-mediated angiogenesis. In a murine model of hindlimb ischemia, daily oral fenofibrate treatment restored diabetes-impaired blood flow recovery, foot movement, hindlimb capillary density, vessel diameter, and vascular endothelial growth factor signaling to nondiabetic levels in both wild-type and PPARα-knockout mice, indicating that these fenofibrate effects are largely PPARα independent. In vitro, fenofibric acid (FFA) rescued high glucose-induced (25 mmol/L) impairment of endothelial cell migration, tubulogenesis, and survival in a PPARα-independent manner. Interestingly, fenofibrate in vivo and FFA in vitro reversed high glucose-induced expression of thioredoxin-interacting protein (TXNIP), an exquisitely glucose-inducible gene previously identified as a critical mediator of diabetes-related impairment in neovascularization. Conversely, adenoviral overexpression of TXNIP abrogated the restorative effects of FFA on high glucose-impaired endothelial cell function in vitro, indicating that the effects of FFA are mediated by TXNIP. We conclude that fenofibrate rescues diabetic impairment in ischemia-mediated angiogenesis, in large part, by PPARα-independent regulation of TXNIP. These findings may therefore explain the reduction in amputations seen in patients with diabetes treated with fenofibrate., (© 2019 by the American Diabetes Association.)

Bone marrow-derived progenitor cell-mediated vasculogenesis is a key process for vascular repair and regeneration. However, the role of androgens in the mechanism of ischemia-induced vasculogenesis remains unclear. In this study, a gender-mismatch murine bone marrow transplant model was used to allow tissue tracking of transplanted cells. Bone marrow from 2-month-old male mice was transplanted into irradiated age-matched female recipients. Following the transplantation, ovariectomized female recipients were subjected to unilateral hindlimb ischemia and immediately implanted with either dihydrotestosterone (DHT) or placebo pellets. Laser Doppler perfusion imaging revealed that DHT significantly augmented blood flow recovery, with increased capillary density compared to placebo-treated female recipient controls. Flow cytometry analysis showed that DHT modulated vasculogenesis by increasing Sca1+/CXC4+ progenitor cell production in bone marrow and spleen and enhancing cell mobilization in circulating blood following hindlimb ischemia. Bone marrow cell homing was examined by detecting expression levels of male-specific SRY gene in the ischemic female tissues. DHT treatment promoted bone marrow cell homing to ischemic tissue shown by significantly higher SRY expression compared to placebo-treated females as well as reduced apoptotic features in DHT-treated females, including increased Bcl-2 expression, reduced Bax levels and decreased TUNEL staining. In conclusion, the gender-mismatched bone marrow transplant study shows that androgens directly enhance bone marrow cell-mediated vasculogenesis that contributes to ischemia-induced neovascularization., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Mesenchymal stem cells (MSCs) reportedly exist in a vascular niche occupying the outer adventitial layer. However, these cells have not been well characterized in vivo in medium- and large-sized arteries in humans, and their potential pathological role is unknown. To address this, healthy and diseased arterial tissues were obtained as surplus surgical specimens and freshly processed. We identified that CD90 marks a rare adventitial population that co-expresses MSC markers including PDGFRα, CD44, CD73, and CD105. However, unlike CD90, these additional markers were widely expressed by other cells. Human adventitial CD90+ cells fulfilled standard MSC criteria, including plastic adherence, spindle morphology, passage ability, colony formation, and differentiation into adipocytes, osteoblasts, and chondrocytes. Phenotypic and transcriptomic profiling, as well as adoptive transfer experiments, revealed a potential role in vascular disease pathogenesis, with the transcriptomic disease signature of these cells being represented in an aortic regulatory gene network that is operative in atherosclerosis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing evidence indicates that androgens regulate ischemia-induced neovascularization. However, the role of genomic androgen action mediated by androgen receptor (AR), a ligand-activated nuclear transcription factor, remains poorly understood. Using an AR knockout (KO) mouse strain that contains a transcriptionally inactive AR (AR Δex3 KO), we examined the role of AR genomic function in modulating androgen-mediated augmentation of ischemia-induced neovascularization. Castrated wild-type (AR WT ) and AR Δex3 KO mice were implanted with 5α-dihydrotestosterone (DHT) or placebo pellets after hindlimb ischemia (HLI). DHT modulation of angiogenesis and vasculogenesis, key processes for vascular repair and regeneration, was examined. Laser Doppler perfusion imaging revealed that DHT enhanced blood flow recovery in AR WT mice post-HLI. In AR WT mice, DHT enhanced angiogenesis by down-regulating prolyl hydroxylase 2 and augmenting hypoxia-inducible factor-1α (HIF-1α) levels in the ischemic tissues post-HLI. DHT also enhanced the production and mobilization of Sca1+/CXCR4+ progenitor cells in the bone marrow (BM) and circulating blood, respectively, in AR WT mice. By contrast, DHT-mediated enhancement of blood flow recovery was abrogated in AR Δex3 KO mice. DHT modulation of HIF-1α expression was attenuated in AR Δex3 KO mice. DHT-induced HIF-1α transcriptional activity and DHT-augmented paracrine-mediated endothelial cell tubule formation were attenuated in fibroblasts isolated from AR Δex3 KO mice in vitro. Furthermore, DHT-induced augmentation of Sca1+/CXCR4+ progenitor cell production and mobilization was absent in AR Δex3 KO mice post-HLI. BM transplantation revealed that ischemia-induced mobilization of circulating progenitor cells was abolished in recipients of AR Δex3 KO BM. Together, these results indicate that androgen-mediated augmentation of ischemia-induced neovascularization is dependent on genomic AR transcriptional activation.

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely β(1) and β(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The β(1) and β(3) integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, β(1) and β(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, β(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in β(1) and β(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, β(1) and β(3) integrin disassembly from focal adhesions and the increase in β(3) integrin apically are key components of hormonally regulated endometrial receptivity.