Mei Mei Wong

Monocytes Induce STAT3 Activation in Human Mesenchymal Stem Cells to Promote Osteoblast Formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.

Regulation of MicroRNA-155 in Atherosclerotic Inflammatory Responses by Targeting MAP3K10

Aims Accumulating evidence suggest that numerous microRNAs (miRNAs) play important roles in cell proliferation, apoptosis, and differentiation, as well as various diseases that accompany inflammatory responses. Inflammation is known to be a major contributor to atherogenesis. Previous studies provide promising evidence in support of the role of miRNAs in cardiovascular disease. However, mechanistic data on these small molecules in atherosclerosis (AS) are still missing. The present study aims to investigate the potential role of miRNAs in AS. Methods and Results The miRNA transcriptase was verified by TaqMan real-time polymerase chain reaction assay. Thoracic aorta samples were obtained from Apolipoprotein E knockout mice, and plasma samples were from coronary artery disease (CAD) patients. The results showed that the miR-155 level was the most significantly elevated both in AS mice and CAD patients relative to the normal control. The functional role of miR-155 in the atherosclerotic path physiological process was also observed in vivo and in vitro. The observations suggested that miR-155 is a part of a negative feedback loop, which down-modulates inflammatory cytokine production and decreases AS progression. miR-155 was also found to mediate the inflammatory response and mitogen-activated protein kinase (MAPK) pathway by targeting mitogen-activated protein kinase kinase kinase 10. Conclusions miR-155 contributes to the prevention of AS development and progression. It may also be involved in the posttranscriptional regulation of the inflammatory response and MAPK pathway by targeting mitogen-activated protein kinase kinase kinase 10.

Adventitial Stem Cells in Vein Grafts Display Multilineage Potential That Contributes to Neointimal Formation

Objective—This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. Approach and Results—A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1–positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1–positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1–positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1–positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E–deficient mice, which contributed to ≈30% of neointimal SMCs. Conclusions—We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.

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.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal–Regulated Kinase/β-Catenin Signaling Pathway

Objective—Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. Approach and Results—Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22&agr; and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal–regulated kinase 1/2 activation that lead to &bgr;-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal–regulated kinase 1/2, or &bgr;-catenin attenuated sirolimus-induced SM-22&agr; promoter activation and SMC differentiation. Conclusions—These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal–regulated kinase/&bgr;-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.

Progenitor Cells in Arteriosclerosis: Good or Bad Guys?

Accumulating evidence indicates that the mobilization and recruitment of circulating or tissue-resident progenitor cells that give rise to endothelial cells (ECs) and smooth muscle cells (SMCs) can participate in atherosclerosis, neointima hyperplasia after arterial injury, and transplant arteriosclerosis. It is believed that endothelial progenitor cells do exist and can repair and rejuvenate the arteries under physiologic conditions; however, they may also contribute to lesion formation by influencing plaque stability in advanced atherosclerotic plaque under specific pathologic conditions. At the same time, smooth muscle progenitors, despite their capacity to expedite lesion formation during restenosis, may serve to promote atherosclerotic plaque stabilization by producing extracellular matrix proteins. This profound evidence provides support to the hypothesis that both endothelial and smooth muscle progenitors may act as a double-edged sword in the pathogenesis of arteriosclerosis. Therefore, the understanding of the regulatory networks that control endothelial and smooth muscle progenitor differentiation is undoubtedly fundamental both for basic research and for improving current therapeutic avenues for atherosclerosis. We update the progress in progenitor cell study related to the development of arteriosclerosis, focusing specifically on the role of progenitor cells in lesion formation and discuss the controversial issues that regard the origins, frequency, and impact of the progenitors in the 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 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.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α–Mediated Nuclear Factor-κB Activation

Objective— Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. Approach and Results— We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-&agr; (TNF-&agr;) via TNF-&agr; receptor 1 and canonical nuclear factor-&kgr;B activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-&agr; receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-&agr;. Furthermore, TNF-&agr;–mediated EC differentiation was driven by direct binding of nuclear factor-&kgr;B (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-&agr;. The lack of TNF-&agr; in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Conclusions— Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-&agr;–mediated TNF-&agr; receptor 1 and nuclear factor-&kgr;B activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Vascular Stem/Progenitor Cell Migration Induced by Smooth Muscle Cell-Derived Chemokine (C-C Motif) Ligand 2 and Chemokine (C-X-C motif) Ligand 1 Contributes to Neointima Formation

Recent studies have shown that Sca‐1+ (stem cell antigen‐1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca‐1+ progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC‐derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C‐C motif) ligand 2) and CXCL1 (chemokine (C‐X‐C motif) ligand 1), and their corresponding receptors on Sca‐1+ progenitors, CCR2 (chemokine (C‐C motif) receptor 2) and CXCR2 (chemokine (C‐X‐C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca‐1+ progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca‐1+ progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire‐injured mouse femoral arteries, a large proportion of GFP‐Sca‐1+‐cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post‐operation. Interestingly, Sca‐1+ progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2−/− mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016;34:2368–2380

Vascular Stem/Progenitor Cell Migration Induced by SMC‐derived CCL2 and CXCL1 Contributes to Neointima Formation

Recent studies have shown that Sca‐1+ (stem cell antigen‐1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca‐1+ progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC‐derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C‐C motif) ligand 2) and CXCL1 (chemokine (C‐X‐C motif) ligand 1), and their corresponding receptors on Sca‐1+ progenitors, CCR2 (chemokine (C‐C motif) receptor 2) and CXCR2 (chemokine (C‐X‐C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca‐1+ progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca‐1+ progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire‐injured mouse femoral arteries, a large proportion of GFP‐Sca‐1+‐cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post‐operation. Interestingly, Sca‐1+ progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2−/− mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016;34:2368–2380