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Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(β)—two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection—but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09823-4.

Islet transplantation has been established as a viable treatment modality for type 1 diabetes. However, the side effects of the systemic immunosuppression required for patients often outweigh its benefits. Here, we engineer programmed death ligand-1 and cytotoxic T lymphocyte antigen 4 immunoglobulin fusion protein–modified mesenchymal stromal cells (MSCs) as accessory cells for islet cotransplantation. The engineered MSCs (eMSCs) improved the outcome of both syngeneic and allogeneic islet transplantation in diabetic mice and resulted in allograft survival for up to 100 days without any systemic immunosuppression. Immunophenotyping revealed reduced infiltration of CD4+or CD8+T effector cells and increased infiltration of T regulatory cells within the allografts cotransplanted with eMSCs compared to controls. The results suggest that the eMSCs can induce local immunomodulation and may be applicable in clinical islet transplantation to reduce or minimize the need of systemic immunosuppression and ameliorate its negative impact.

The success of vascular progenitor cell transplantation to treat myocardial infarction (MI) is primarily limited by the low engraftment of delivered cells due to a washout effect during myocardium contraction. A clinically applicable biomaterial to improve cell retention is arguably needed to enable optimization of intramyocardial cell delivery. Here, we developed a novel therapeutic cell delivery method for MI treatment based on a photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. A combination of human vascular progenitor cells (endothelial progenitors and mesenchymal stem cells) with the capacity to form functional vasculatures after transplantation, were injected with a rapid in-situ photopolymerization approach into the infarcted zone of mouse hearts. Our approach significantly improved acute cell retention and achieved a long-term beneficial post-MI cardiac healing, including stabilizing cardiac functions, preserving viable myocardium, and preventing cardiac fibrosis. Furthermore, the engrafted vascular cells polarized recruited bone marrow-derived neutrophils toward a non-inflammatory phenotype via TGFβ signaling, establishing a pro-regenerative microenvironment. Depletion of neutrophils canceled the therapeutic benefits produced by cell delivery in the ischemic hearts, indicating that the non-inflammatory, pro-regenerative neutrophils were indispensable mediators of cardiac remodeling. In summary, our novel GelMA hydrogel-based intramyocardial vascular cell delivery approach has the potential to improve the treatment of acute MI.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Hemophilia A (HA) is a bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatments are based on regular infusions of FVIII concentrates throughout a patient’s life. Alternatively, viral gene therapies that directly deliver F8 in vivo have shown preliminary successes. However, hurdles remain, including lack of infection specificity and the inability to deliver the full-length version of F8 due to restricted viral cargo sizes. Here, we developed an alternative nonviral ex vivo gene-therapy approach that enables the overexpression of full-length F8 in patients’ endothelial cells (ECs). We first generated HA patient–specific induced pluripotent stem cells (HA-iPSCs) from urine epithelial cells and genetically modified them using a piggyBac DNA transposon system to insert multiple copies of full-length F8. We subsequently differentiated the modified HA-iPSCs into competent ECs with high efficiency, and demonstrated that the cells (termed HA-FLF8-iECs) were capable of producing high levels of FVIII. Importantly, following subcutaneous implantation into immunodeficient hemophilic (SCID-f8ko) mice, we demonstrated that HA-FLF8-iECs were able to self-assemble into vascular networks, and that the newly formed microvessels had the capacity to deliver functional FVIII directly into the bloodstream of the mice, effectively correcting the clotting deficiency. Moreover, our implant maintains cellular confinement, which reduces potential safety concerns and allows effective monitoring and reversibility. We envision that this proof-of-concept study could become the basis for a novel autologous ex vivo gene-therapy approach to treat HA.

Objective— To determine the role of a cytokine-like protein DKK3 (dikkopf-3) in directly transdifferentiating fibroblasts into endothelial cells (ECs) and the underlying mechanisms. Approach and Results— DKK3 overexpression in human fibroblasts under defined conditions for 4 days led to a notable change in cell morphology and progenitor gene expression. It was revealed that these cells went through mesenchymal-to-epithelial transition and subsequently expressed KDR (kinase insert domain receptor) at high levels. Further culture in EC defined media led to differentiation of these progenitors into functional ECs capable of angiogenesis both in vitro and in vivo, which was regulated by the VEGF (vascular endothelial growth factor)/miR (microRNA)-125a-5p/Stat3 (signal transducer and activator of transcription factor 3) axis. More importantly, fibroblast-derived ECs showed the ability to form a patent endothelium-like monolayer in tissue-engineered vascular grafts ex vivo. Conclusions— These data demonstrate that DKK3 is capable of directly differentiating human fibroblasts to functional ECs under defined media and provides a novel potential strategy for endothelial regeneration.

Visual Abstract, Highlights • EFE is a congenital cardiac pathology contributing to increased morbidity and mortality. The pathologic triggers of EFE remain to be characterized. • To determine whether abnormal flow promotes EFE development, we used in vivo neonatal rodent surgical models and an in vitro model using human primary endocardial cells • We established novel surgical model with flow profiles seen in patients that develop EFE. Static and turbulent flow conditions promoted EFE development in neonatal rodent hearts. • Losartan treatment is shown to significantly ameliorate EFE progression and decreases mRNA and protein expression of EndoMT markers in neonatal rodent hearts. • RNAseq analysis of human endocardial cells subjected to different flow conditions show that normal flow suppresses gene expression critical for mesenchymal differentiation and Notch signaling., Summary Endocardial fibroelastosis (EFE) is defined by fibrotic tissue on the endocardium and forms partly through aberrant endothelial-to-mesenchymal transition. However, the pathologic triggers are still unknown. In this study, we showed that abnormal flow induces EFE partly through endothelial-to-mesenchymal transition in a rodent model, and that losartan can abrogate EFE development. Furthermore, we translated our findings to human endocardial endothelial cells, and showed that laminar flow promotes the suppression of genes associated with mesenchymal differentiation. These findings emphasize the role of flow in promoting EFE in endocardial endothelial cells and provide a novel potential therapy to treat this highly morbid condition.

Introduction: Endocardial Fibroelastosis (EFE) is a condition defined by the formation of fibrotic tissue on the endocardium, stunting cardiac development. The presence of EFE restricts diastolic compliance, leading to systolic and diastolic heart failure, and contributing to higher morbidity and mortality, particularly in Hypoplastic Left Heart Syndrome. While research suggests that EFE forms through aberrant endothelial-to-mesenchymal transition (EndoMT), the trigger of its formation is still speculative. Anecdotally, however, we have observed EFE development in patients with abnormal left ventricular flow. Objective: Our objective is to study whether the absence of normal flow induces EndoMT in neonatal endocardium and promotes EFE development, and whether losartan treatment, which can decrease TGFβ production to impair EndoMT, can abrogate EFE formation. Methods and Results: Using a rodent model of EFE, we establish three flow conditions in neonatal hearts: Normal Flow (NF), Static/no flow (S), and Regurgitant Flow (RF). Histologic analysis demonstrates EFE development in S and RF conditions, but not in NF conditions. Using qPCR, Western Blot Assay, and immunohistochemistry, we demonstrate that S and RF significantly increase mRNA and protein expression of EndoMT markers (αSMA, SNAIL), and EndoMT-related signaling pathways (TGFβ, NOTCH1) whereas NF conditions do not. With losartan treatment, we observe a significant decrease in EFE in neonatal hearts documented by histology and decreased mRNA and protein expression of EndoMT markers. To determine whether our findings are translatable to humans, human endocardial endothelial cells were isolated from left ventricular tissue and subjected to two flow conditions: static and laminar shear stress (LSS). By RNAseq analysis, we demonstrate that laminar shear stress suppresses the expression of genes critical for mesenchymal differentiation and Notch signaling, which are both associated with EndoMT progression. Conclusions: Our data demonstrates that the absence of normal flow provides an environment that promotes EFE development through activated EndoMT. Further, we identify losartan as a potential therapy for EFE, as it is able to abrogate EFE development in our animal model.

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and − 2 in ECFCs. Graphical Abstract

In article number 2100070, Juan M. Melero-Martin, Ruei-Zeng Lin, and co-workers present a biphasic scaffold that allows self-sustained endochondral ossification. Following seeding with human vascular and mesenchymal stem cells, this biomimetic material enables the rapid generation of osteovascular niches and ossification centers, leading to the formation of vascularized bone tissue in vivo.

Vascular remodeling is a complex and dynamic pathological process engaging many different cell types that reside within the vasculature. Mesenchymal stromal/stem cells (MSCs) refer to a heterogeneous cell population with the plasticity to differentiate toward multiple mesodermal lineages. Various types of MSC have been identified within the vascular wall that actively contribute to the vascular remodeling process such as atherosclerosis. With the advances of genetic mouse models, recent findings demonstrated the crucial roles of MSCs in the progression of vascular diseases. This review aims to provide an overview on the current knowledge of the characteristics and behavior of vascular resident MSCs under quiescence and remodeling conditions, which may lead to the development of novel therapeutic approaches for cardiovascular diseases.

Regeneration of large bones remains a challenge in surgery. Recent developmental engineering efforts aim to recapitulate endochondral ossification (EO), a critical step in bone formation. However, this process entails the condensation of mesenchymal stem cells (MSCs) into cartilaginous templates, which requires long-term cultures and is challenging to scale up. Here, we develop a biomimetic scaffold that allows rapid and self-sustained EO without initial hypertrophic chondrogenesis. The design comprises a porous chondroitin sulfate (CS) cryogel decorated with whitlockite calcium phosphate nanoparticles, and a soft hydrogel occupying the porous space. This composite scaffold enables human endothelial colony-forming cells (ECFCs) and MSCs to rapidly assemble into osteovascular niches in immunodeficient mice. These niches contain ECFC-lined blood vessels and perivascular MSCs that differentiate into RUNX2(+)OSX(+) pre-osteoblasts after one week in vivo. Subsequently, multiple ossification centers are formed, leading to de novo bone tissue formation by eight weeks, including mature human OCN(+)OPN(+) osteoblasts, collagen-rich mineralized extracellular matrix, hydroxyapatite, osteoclast activity, and gradual mechanical competence. The early establishment of blood vessels are essential, and grafts that do not contain ECFCs fail to produce osteovascular niches and ossification centers. Our findings suggest a novel bioengineering approach to recapitulate EO in the context of human bone regeneration.

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it would be of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium. Indeed, recent achievements of cell reprogramming to convert somatic cells to other cell types provide new powerful approaches to study endothelial regeneration. Based on progress in the research field, the present review aims to explore the strategies and mechanisms of generating endothelial cells through reprogramming from somatic cells, and to examine what this means for the potential application of cell therapy in the clinic.

Objectives: Tissue engineered vascular grafts with long-term patency are in great need in the clinics, for which smooth muscle cells (SMCs) are essential. Recently several laboratories have established methods to obtain SMCs from embryonic stem cells or iPS cells. However, the mechanisms of stem cell differentiation are not fully understood. And also it is hard to obtain a large number of SMCs with higher purity. To solve these issues, we take the advantage of mesenchymal stem cells (MSCs) from human umbilical cord that were cultivated and differentiated in vitro. Methods: MSCs from human umbilical cord were cultivated and differentiated to vascular lineages. Signal pathways involved in stem cell differentiation were studied using Western blot, siRNA, PCR, immunoprecipation and transfection. For vascular tissue engineering, stem cell-derived SMCs were applied to decelularized scaffold to create the vessels in a Bioreactor. Results: In response to TGFβ1 stimulation, MSCs were abundantly expressing SMC markers including αSMA, SM22, calponin and SMMHC at the gene and protein levels. Functionally, differentiated SMCs displayed a contraction ability in vitro and formed vessels in matrigel plug assay SCID mice. Micro-RNA (miR) array analysis showed the upregulation of miR-503 and the downregulation of miR-222-5p at early time points after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. Mechanistically, miR-503 was demonstrated to promote SMC differentiation through directly targeting Smad7, a negative regulator of TGFβ1 Smad-related signaling pathways. The expression of miR-503 was Smad4-dependent and Smad4 was enriched at the promoter region of miR-503. In addition, miR-222-5p inhibited SMC differentiation through targeting ROCK2 and αSMA 3’-UTR. Finally, SMCs differentiated from stem cells exhibited the ability to migrate into decellularized mouse aorta and gave rise to vascular graft with smooth muscle layer. Conclusions: We established a method to produce a large number of human SMCs from MSCs, which demonstrated the potential of utilizing the cells to generate vascular grafts. And we provide the mechanistic data demonstrating the impact of miRNA-503/222-5p on TGFβ1-mediated signaling in SMC differentiation from stem cells.

Supplemental Digital Content is available in the text., Objective— Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. Approach and Results— We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control. Conclusions— These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.

In recent years, MSCs have emerged as a promising therapeutic cell type in regenerative medicine. They hold great promise for treating cardiovascular diseases, such as myocardial infarction and limb ischemia. MSCs may be utilized in both cell-based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits to patients. The efficacy of MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Increasing our understanding of the mechanisms of MSC involvement in vascular regeneration will be beneficial in boosting present therapeutic approaches and developing novel ones to treat cardiovascular diseases. In this review, we aim to summarize current progress in characterizing the in vivo identity of MSCs, to discuss mechanisms involved in cell-based therapy utilizing MSCs, and to explore current and future strategies for vascular regeneration.

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 β-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/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

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.

Introduction Endothelial damage/dysfunction is a key event in cardiovascular pathological changes and development of ischaemic vascular disease, which is often followed by smooth muscle cell (SMC) proliferation and accumulation. If we could arbitrarily convert SMCs into endothelial cells (ECs), they may represent a promising new approach to achieve therapeutic angiogensis of ischaemic area. To test this hypothesis, the aim of the present study is to transdifferentiate human SMCs into functional ECs. Methods and results Human SMCs were first reprogrammed for 4 days to obtain a partially converted state, i.e. vascular progenitor, by introducing four transcription factors: Oct4, Sox2, Klf4 and c-Myc . These vascular progenitors were then subjected to defined media and culture conditions to induce endothelial lineage differentiation. The differentiated cells expressed EC markers such as CD31, CD144, and eNOS at the mRNA and protein level. Next, CD34 positive cells were selected from the heterogeneous population of vascular progenitors and further cultured in endothelial inducing conditions. The CD34+ cells were able to give rise to a more homogenous endothelial-like population in higher efficiency. The converted ECs displayed typical endothelial functional properties including acetylated-LDL uptake and tube formation in vitro and in vivo. More importantly, these ECs-derived from human SMCs exhibited therapeutic angiogenesis capacity which improved blood flow recovery when applied in the murine ischaemic hindlimb model. To explore the mechanisms involved in SMCs to ECs transdifferentiation, whole mRNAs from different reprogrammed cells were analysed by RNA-Sequencing techniques. Comprehensive analysis indicated that mesenchymal-to-epithelial transition was requisite to initiate SMCs reprogramming to vascular progenitor. Furthermore, it was demonstrated that hairy and enhancer of split 5 (HES5), a member of a family of basic helix-loop-helix transcriptional factor of the Notch signalling pathway, regulated progenitors to endothelial lineage differentiation. Conclusions In this study, we provided the first evidence of converting human SMCs towards functional EC lineage involving the mechanisms of mesenchymal-to-epithelial transition and the Notch signalling pathway. Furthermore, the converted ECs displayed a therapeutic capacity of angiogenesis in a murine ischaemic model.

Background and Purpose:Vessel graft failure is typically associated with arteriosclerosis, in which endothelial dysfunction/damage is a key event. Resveratrol has been shown to possess cardioprotective capacity and to reduce atherosclerosis. We aimed to study the influence of resveratrol on the behavior of resident stem cells that may contribute to graft arteriosclerosis.Experimental Approach:Vascular resident progenitor cells and embryonic stem cells were treated with resveratrol under differentiating conditions and endothelial markers expression was evaluated. Expression of miR-21 and β-catenin was also tested and exogenously modified. Effects of resveratrol treatment in an ex vivo re-endothelialization model and on mice undergone vascular graft were evaluated.Key Results:Resveratrol induced expression of endothelial markers such as CD31, VE-cadherin and eNOS in both progenitor and stem cells. We demonstrated that resveratrol significantly reduced miR-21 expression, which in turn reduced Akt phosphorylation. This signal cascade diminished the amount of nuclear β-catenin, inducing endothelial marker expression and increasing tube-like formation by progenitor cells. Both the inhibition of miR-21 and the knockdown of β-catenin were able to recapitulate the effect of resveratrol application. Ex vivo, progenitor cells treated with resveratrol produced better endothelialization of the decellularized vessel. Finally, in a mouse model of vessel graft, a resveratrol-enhanced diet was able to reduce lesion formation.Conclusions and Implications:We provide the first evidence that oral administration of resveratrol can reduce neointimal formation in a model of vascular graft and elucidated the underpinning miR-21/Akt/β-catenin dependent mechanism. These findings may support the beneficial effect of resveratrol supplementation for graft failure prevention.

Background Vessel graft failure is typically associated neointimal formation and arteriosclerosis, in which endothelial dysfunction/damage is a key event.1 Resveratrol has been shown to possess cardioprotective capacity and to reduce atherosclerosis, through its anti-oxidant and anti-apoptotic properties.2–4 However, it is unknown whether it influences the behavior of resident stem cells in the vessel wall leading to the development of arteriosclerosis. Methods and results In the present study, the mouse model of vein grafts was established by grafting vena cava to carotid artery using a cuff technique. ApoE knockout animals were randomly assigned to two groups, one receiving a diet enriched with resveratrol and one control group on a normal diet. It was found that neointimal lesions 4 weeks postoperatively were significantly reduced in resveratrol-treated group compared to untreated controls. Immunostaining for cell components in the grafts revealed the presence of Sca-1+ progenitor cells in the lesional adventitia and neointima of vein grafts. Subsequently, Sca-1+ cells from 4-week vein grafts were cultivated and isolated. Interestingly, stem/progenitor cell differentiation into endothelial lineage was markedly increased by treatment with resveratrol in vitro. We investigated the mechanism involved in the resveratrol-induced progenitor cell differentiation and identified miR-21 as a target of resveratrol. We demonstrated that resveratrol significantly reduced miR-21 expression during endothelial differentiation of progenitor cells, which in turn reduced Akt phosphorylation. This signal cascade diminished the amount of nuclear β-catenin, ultimately inducing endothelial marker expression and tube-like formation capacity in stem/progenitor cells. Both the inhibition of miR-21 and the knockdown of β-catenin were able to resemble the effect of resveratrol application, i.e. reducing endothelial differentiation. Finally, the effect of resveratrol on progenitor cell differentiation was blunted by the overexpression of miR-21. Conclusion We provide the first evidence that oral administration of resveratrol can reduce neointimal formation in an animal model of vein graft, by inducing re-endothelialization through progenitor cell differentiation. We established that the mechanism involved is miR-21/Akt/β-catenin dependent. These findings might contribute to explain the beneficial effect of red wine consumption on vascular disease.

Background Emerging evidence indicates that the mobilization and recruitment of circulating or tissue-resident stem/progenitor cells give rise to smooth muscle cells (SMCs) which participate in numerous cardiovascular diseases. Understanding the regulatory mechanisms that control SMC differentiation and their recruitment from vascular progenitors is essential for stem cell therapy for vascular diseases and regenerative medicine. Hyaluronan (HA) is a linear glycosominoglycan and ubiquitous component of the intercellular matrix; the identification of HA as a critical component of the stem cell niche led us to investigate its role in regulating stem cell differentiation. Methods and Results Stem cells were seeded on collagen-IV-coated flasks and cultured in differentiation medium to induce SMC differentiation as demonstrated by the expression of SMC-specific genes smooth muscle α-actin, calponin, SM22-α and smooth muscle myosin heavy chain. These phenotypic changes coincided with dynamic changes in HA synthesis and organization. It was demonstrated that SMC differentiation was associated with increased HA synthase (HAS)2 expression, extracellular HA synthesis and loss of pericellular HA. Inhibition of HA synthesis by hyaluronidase, 4-methylumbelliferone (4MU), or specific gene silencing of HAS2 led to abrogation of SMC marker expression. Furthermore, It was found that SMC differentiation was coupled to phosphorylation of the EGF-receptor and ERK1/2, this was shown to be HA-dependent as pre-treatment with 4MU prevented downstream events. Paradoxically, when applied exogenously as a culture medium supplement, low molecular weight HA promotes, whereas high molecular weight HA attenuates SMC differentiation. Subsequently, using a bioreactor system, low molecular weight HA was circulated through a decellurised vessel scaffold ex vivo. In contrast to differentiation medium alone the addition of low molecular weight HA promoted migration of stem cells seeded around the outside of the vessel into the media and their differentiation into SMCs, which coincided with localisation of HA and its receptor CD44. Conclusions We demonstrate for the first time that endogenously produced HA is critical for SMC differentiation from stem cells, via activation of EGF-R and ERK1/2. The prospect of maintaining stemness or inducing differentiation in stem cells by manipulation of HA concentration/molecular weight, or by targeting genes activated by HA, may provide new therapies for vascular disease.