Clopidogrel-loaded vascular grafts prepared using digital light processing 3D printing.

The leading cause of death worldwide and a significant factor in decreased quality of life are the cardiovascular diseases. Endovascular operations like angioplasty, stent placement, or atherectomy are often used in vascular surgery to either dilate a narrowed blood artery or remove a blockage. As an alternative, a vascular transplant may be utilised to replace or bypass a dysfunctional or blocked blood vessel. Despite the advancements in endovascular surgery and its popularisation over the past few decades, vascular bypass grafting remains prevalent and is considered the best option for patients in need of long-term revascularisation treatments. Consequently, the demand for synthetic vascular grafts composed of biocompatible materials persists. To address this need, biodegradable clopidogrel (CLOP)-loaded vascular grafts have been fabricated using the digital light processing (DLP) 3D printing technique. A mixture of polylactic acid-polyurethane acrylate (PLA-PUA), low molecular weight polycaprolactone (L-PCL), and CLOP was used to achieve the required mechanical and biological properties for vascular grafts. The 3D printing technology provides precise detail in terms of shape and size, which lead to the fabrication of customised vascular grafts. The fabricated vascular grafts were fully characterised using different techniques, and finally, the drug release was evaluated. Results suggested that the performed 3D-printed small-diameter vascular grafts containing the highest CLOP cargo (20% w/w) were able to provide a sustained drug release for up to 27 days. Furthermore, all the CLOP-loaded 3D-printed materials resulted in a substantial reduction of the platelet deposition across their surface compared to the blank materials containing no drug. Haemolysis percentage for all the 3D-printed samples was lower than 5%. Moreover, 3D-printed materials were able to provide a supportive environment for cellular attachment, viability, and growth. A substantial increase in cell growth was detected between the blank and drug-loaded grafts.

3D-printed reservoir-type implants containing poly(lactic acid)/poly(caprolactone) porous membranes for sustained drug delivery.

Implantable drug delivery systems are an interesting alternative to conventional drug delivery systems to achieve local or systemic drug delivery. In this work, we investigated the potential of fused-deposition modelling to prepare reservoir-type implantable devices for sustained drug delivery. An antibiotic was chosen as a model molecule to evaluate the potential of this type of technology to prepare implants on-demand to provide prophylactic antimicrobial treatment after surgery. The first step was to prepare and characterize biodegradable rate-controlling porous membranes based on poly(lactic acid) (PLA) and poly(caprolactone) (PCL). These membranes were prepared using a solvent casting method. The resulting materials contained different PLA/PCL ratios. Cylindrical implants were 3D-printed vertically on top of the membranes. Tetracycline (TC) was loaded inside the implants and drug release was evaluated. The results suggested that membranes containing a PLA/PCL ratio of 50/50 provided drug release over periods of up to 25 days. On the other hand, membranes containing lower PCL content did not show a porous structure and accordingly the drug could not permeate to the same extent. The influence of different parameters on drug release was evaluated. It was established that film thickness, drug content and implant size are critical parameters as they have a direct influence on drug release kinetics. In all cases the implants were capable of providing drug release for at least 25 days. The antimicrobial properties of the implants were evaluated against E. coli and S. aureus. The resulting implants showed antimicrobial properties at day 0 and even after 21 days against both type of microorganisms. Finally, the biocompatibility of the implants was evaluated using endothelial cells. Cells exposed to implants were compared with a control group. There were no differences between both groups in terms of cell proliferation and morphology.

RNA-Binding Proteins Hold Key Roles in Function, Dysfunction, and Disease.

RNA-binding proteins (RBPs) are multi-faceted proteins in the regulation of RNA or its RNA splicing, localisation, stability, and translation. Amassing proof from many recent and dedicated studies reinforces the perception of RBPs exerting control through differing expression levels, cellular localization and post-transcriptional alterations. However, since the regulation of RBPs is reliant on the micro-environment and events like stress response and metabolism, their binding affinities and the resulting RNA-RBP networks may be affected. Therefore, any misregulation and disruption in the features of RNA and its related homeostasis can lead to a number of diseases that include diabetes, cardiovascular disease, and other disorders such as cancer and neurodegenerative diseases. As such, correct regulation of RNA and RBPs is crucial to good health as the effect RBPs exert through loss of function can cause pathogenesis. In this review, we will discuss the significance of RBPs and their typical function and how this can be disrupted in disease.

Endothelial Cells Derived From Patients With Diabetic Macular Edema Recapitulate Clinical Evaluations of Anti-VEGF Responsiveness Through the Neuronal Pentraxin 2 Pathway.

Diabetic macular edema (DME) remains a leading cause of vision loss worldwide. DME is commonly treated with intravitreal injections of vascular endothelial growth factor (VEGF)-neutralizing antibodies. VEGF inhibitors (anti-VEGFs) are effective, but not all patients fully respond to them. Given the potential side effects, inconvenience, and high cost of anti-VEGFs, identifying who may not respond appropriately to them and why is essential. Herein we determine first the response to anti-VEGFs, using spectral-domain optical coherence tomography scans obtained from a cohort of patients with DME throughout the 1st year of treatment. We found that fluid fully cleared at some time during the 1st year in 28% of eyes ("full responders"); fluid cleared only partly in 66% ("partial responders"); and fluid remained unchanged in 6% ("nonresponders"). To understand this differential response, we generated induced pluripotent stem cells (iPSCs) from full responders and nonresponders, from subjects with diabetes but no DME, and from age-matched volunteers without diabetes. We differentiated these iPSCs into endothelial cells (iPSC-ECs). Monolayers of iPSC-ECs derived from patients with diabetes showed a marked and prolonged increase in permeability upon exposure to VEGF; the response was significantly exaggerated in iPSC-ECs from nonresponders. Moreover, phosphorylation of key cellular proteins in response to VEGF, including VEGFR2, and gene expression profiles, such as that of neuronal pentraxin 2, differed between full responders and nonresponders. In this study, iPSCs were used in order to predict patients' responses to anti-VEGFs and to identify key mechanisms that underpin the differential outcomes observed in the clinic. This approach identified NPTX2 as playing a significant role in patient-linked responses and as having potential as a new therapeutic target for DME.

Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling.

The mortality rate for (cardio)-vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS-ECs) with typical EC characteristics. This research combined iPS cell technologies and next-generation sequencing to acquire an insight into the transcriptional regulation of iPS-ECs. We identified endothelial cell-specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS-ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signaling, opening the horizon for drug screening and cell-based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell-derived ECs from a very small amount of blood through ESM1 signaling, which greatly enhances their functionality and increases their therapeutic potential. Stem Cells 2019;37:226-239.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis.

The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA-binding protein Quaking isoform 5 (QKI-5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA-binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient-specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI-5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI-5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3' UTR of STAT3. Importantly, mouse iPS-ECs overexpressing QKI-5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI-5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI-5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI-5 is induced during EC differentiation from iPSCs. RNA binding protein QKI-5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI-5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). Stem Cells 2017 Stem Cells 2017;35:952-966.

MicroRNA-199a induces differentiation of induced pluripotent stem cells into endothelial cells by targeting sirtuin 1.

The ability to reprogram induced pluripotent stem (iPS) cells from somatic cells may facilitate significant advances in regenerative medicine. MicroRNAs (miRNAs) are involved in a number of core biological processes, including cardiogenesis, hematopoietic lineage differentiation and oncogenesis. An improved understanding of the complex molecular signals that are required for the differentiation of iPS cells into endothelial cells (ECs) may allow specific targeting of their activity in order to enhance cell differentiation and promote tissue regeneration. The present study reports that miR­199a is involved in EC differentiation from iPS cells. Augmented expression of miR­199a was detected during EC differentiation, and reached higher levels during the later stages of this process. Furthermore, miR­199a inhibited the differentiation of iPS cells into smooth muscle cells. Notably, sirtuin 1 was identified as a target of miR­199a . Finally, the ability of miR­199a to induce angiogenesis was evaluated in vitro, using Matrigel plugs assays. This may indicate a novel function for miR­199a as a regulator of the phenotypic switch during vascular cell differentiation. The present study provides support to the notion that with an understanding of the molecular mechanisms underlying vascular cell differentiation, stem cell regenerative therapy may ultimately be developed as an effective treatment for cardiovascular disease.

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway.

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through ß-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/ß-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

MicroRNA-199b Modulates Vascular Cell Fate During iPS Cell Differentiation by Targeting the Notch Ligand Jagged1 and Enhancing VEGF Signaling.

AIMS: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. METHODS AND RESULTS: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis. CONCLUSIONS: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405-1418.

Unspliced X-box-binding protein 1 (XBP1) protects endothelial cells from oxidative stress through interaction with histone deacetylase 3.

It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

Direct reprogramming of adult cells: avoiding the pluripotent state.

The procedure of using mature, fully differentiated cells and inducing them toward other cell types while bypassing an intermediate pluripotent state is termed direct reprogramming. Avoiding the pluripotent stage during cellular conversions can be achieved either through ectopic expression of lineage-specific factors (transdifferentiation) or a direct reprogramming process that involves partial reprogramming toward the pluripotent stage. Latest advances in the field seek to alleviate concerns that include teratoma formation or retroviral usage when it comes to delivering reprogramming factors to cells. They also seek to improve efficacy and efficiency of cellular conversion, both in vitro and in vivo. The final products of this reprogramming approach could be then directly implemented in regenerative and personalized medicine.

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor ß2 (TGF-ß2) pathways.

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-ß2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-ß2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFß-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFß-2, strongly decreased VE-cadherin expression. Furthermore, TGFß-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

Histone deacetylase 3 unconventional splicing mediates endothelial-to-mesenchymal transition through transforming growth factor ß2.

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -ß, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor ß2 (TGFß2) secretion and cleavage. TGFß2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFß2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

Dysfunction of endothelial progenitor cells from smokers and chronic obstructive pulmonary disease patients due to increased DNA damage and senescence.

Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention.

Smooth muscle cells differentiated from reprogrammed embryonic lung fibroblasts through DKK3 signaling are potent for tissue engineering of vascular grafts.

RATIONALE: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. OBJECTIVE: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state. METHODS AND RESULTS: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft. CONCLUSIONS: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.

BACKGROUND: Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. METHODS AND RESULTS: We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3ß phosphorylation, ß-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. CONCLUSIONS: These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis.

Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels.

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

Splicing of histone deacetylase 7 modulates smooth muscle cell proliferation and neointima formation through nuclear ß-catenin translocation.

OBJECTIVE: Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation. METHODS AND RESULTS: We demonstrated that overexpression of unspliced HDAC7 (HDAC7u) could suppress SMC proliferation through downregulation of cyclin D1 and cell cycle arrest, whereas spliced HDAC7 (HDAC7s) could not. Small interfering RNA (siRNA)-mediated knockdown of HDAC7 increased SMC proliferation and induced nuclear translocation of ß-catenin. Additional experiments showed that only HDAC7u could bind to ß-catenin and retain it in the cytoplasm. Reporter gene assay and reverse transcription polymerase chain reaction revealed a reduction of ß-catenin activity in cells overexpressing HDAC7u but not HDAC7s. Deletion studies indicated that the C-terminal region of HDAC7u is responsible for the interaction with ß-catenin. However, the addition of amino acids to the N terminus of HDAC7u disrupted the binding, further strengthening our hypothesis that HDAC7s does not interact with ß-catenin. The growth factor platelet-derived growth factor-BB increased the splicing of HDAC7 while simultaneously decreasing the expression of HDAC7u. Importantly, in an animal model of femoral artery wire injury, we demonstrated that knockdown of HDAC7 by siRNA aggravates neointima formation in comparison with control siRNA. CONCLUSION: Our findings demonstrate that splicing of HDAC7 modulates SMC proliferation and neointima formation through ß-catenin nuclear translocation, which provides a potential therapeutic target in vascular disease.

Role of histone deacetylases in vascular cell homeostasis and arteriosclerosis.

Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups from lysine residues of histone proteins, a modification that results in epigenetic modulation of gene expression. Although originally shown to be involved in cancer and neurological disease, HDACs are also found to play crucial roles in arteriosclerosis. This review summarizes the effects of HDACs and HDAC inhibitors on proliferation, migration, and apoptosis of endothelial and smooth muscle cells. In addition, an updated discussion of HDACs' recently discovered effects on stem cell differentiation and atherosclerosis is provided. Overall, HDACs appear to be promising therapeutic targets for the treatment of arteriosclerosis and other cardiovascular diseases.