Contribution of stem cells to neointimal formation of decellularized vessel grafts in a novel mouse model.

Artificial vessel grafts are often used for the treatment of occluded blood vessels, but neointimal lesions commonly occur. To both elucidate and quantify which cell types contribute to the developing neointima, we established a novel mouse model of restenosis by grafting a decellularized vessel to the carotid artery. Typically, the graft developed neointimal lesions after 2 weeks, resulting in lumen closure within 4 weeks. Immunohistochemical staining revealed the presence of endothelial and smooth muscle cells, monocytes, and stem/progenitor cells at 2 weeks after implantation. Explanted cultures of neointimal tissues displayed heterogeneous outgrowth in stem cell medium. These lesional cells expressed a panel of stem/progenitor markers, including c-kit, stem cell antigen-1 (Sca-1), and CD34. Furthermore, these cells showed clonogenic and multilineage differentiation capacities. Isolated Sca-1(+) cells were able to differentiate into endothelial and smooth muscle cells in response to vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF)-BB stimulation in vitro. In vivo, local application of VEGF to the adventitial side of the decellularized vessel increased re-endothelialization and reduced neointimal formation in samples at 4 weeks after implantation. A population of stem/progenitor cells exists within developing neointima, which displays the ability to differentiate into both endothelial and smooth muscle cells and can contribute to restenosis. Our findings also indicate that drugs or cytokines that direct cell differentiation toward an endothelial lineage may be effective tools in the prevention or delay of restenosis.

Endothelial precursors in vascular repair.

The endothelium is an essential component of the cardiovascular system, playing a vital role in blood vessel formation, vascular homeostasis, permeability and the regulation of inflammation. The integrity of the endothelial monolayer is also critical in the prevention of atherogenesis and as such, restoration of the monolayer is essential following damage or cell death. Over the past decade, data has suggested that progenitor cells from different origins within the body are released into the circulation and contribute to re-endothelialisation. These cells, termed endothelial progenitor cells (EPCs), also gave rise to the theory of new vessel formation within adults (vasculogenesis) without proliferation and migration of mature endothelial cells (angiogenesis). As such, intense research has been carried out identifying how these cells may be mobilised and contribute to vascular repair, either encouraging vasculogenesis into regions of ischemia or the re-endothelialisation of vessels with a dysfunctional endothelium. However, classification and isolation procedures have been a major problem in this area of research and beneficial use for therapeutic application has been controversial. In the present review we focus on the role of EPCs in vascular repair. We also provide an update on EPC classification and discuss autologous stem cell-derived endothelial cell (EC) as a functional source for therapy.

Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease?

The aberrant differentiation of pericytes along the adipogenic, chondrogenic, and osteogenic lineages may contribute to the development and progression of several vascular diseases, including atherosclerosis and calcific vasculopathies. However, the mechanisms controlling pericyte differentiation and, in particular, adipogenic and chondrogenic differentiation are poorly defined. Wnt/beta-catenin signaling regulates cell differentiation during embryonic and postnatal development, and there is increasing evidence that it is involved in vascular pathology. Therefore, this study tested the hypothesis that Wnt/beta-catenin signaling regulates the chondrogenic and adipogenic differentiation of pericytes. We demonstrate that pericytes express several Wnt receptors, including LDL receptor-related proteins 5 and 6, and Frizzled 1 to 4 and 7, 8, and 10, and that Wnt/beta-catenin signaling is stimulated by both Wnt3a and LiCl. Furthermore, induction of Wnt/beta-catenin signaling by LiCl enhances chondrogenesis in pericyte pellet cultures in the presence of transforming growth factor-beta3, as demonstrated by increased Sox-9 expression and glycosaminoglycan accumulation into the matrix. In contrast, transduction of pericytes with a recombinant adenovirus encoding dominant-negative T-cell factor-4 (RAd/dnTCF), which blocks Wnt/beta-catenin signaling, inhibited chondrogenesis, leading to reduced Sox-9 and type II collagen expression and less glycosaminoglycan accumulation. Together, these data demonstrate that transforming growth factor-beta3 induces the chondrogenic differentiation of pericytes by inducing Wnt/beta-catenin signaling and T-cell factor-induced gene transcription. Induction of Wnt/beta-catenin signaling also attenuates adipogenic differentiation of pericytes in both pellet and monolayer cultures, as demonstrated by decreased staining with oil red O and reduced peroxisome proliferator-activated receptor gamma2 expression. This effect was negated by transduction of pericytes with RAd/dnTCF. Together, these results demonstrate that Wnt/beta-catenin signaling inhibits adipogenic and enhances chondrogenic differentiation of pericytes.

Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells.

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.

Vascular repair by endothelial progenitor cells.

Accumulating evidence indicates the impact of endothelial progenitor cells (EPCs) in vascular repair. In patients, the number of EPCs is negatively correlated with the severity of atherosclerosis. In various animal models, transplantation of bone marrow-derived progenitor cells could sufficiently rescue organ function and enhance vascular repair and tissue regeneration. Increase in the number of circulating progenitors, induced by cell transfusion or enhanced mobilization, can also enhance restoration and integrity of the endothelial lining, suppress neointimal formation, and increase blood flow to ischaemic sites. However, the beneficial outcome of EPC infusion very much depends on the growth and differentiation factors within the tissue, cell-to-cell interactions, and the degree of injury. As highlighted by several studies, EPCs derive from different sources including bone marrow and non-bone marrow organs such as the spleen, the functional repair properties of which may vary with the maturation state of the cell. Thus, understanding the molecular mechanisms involved in EPC-repairing processes is essential. In the present review we focus on the role of EPCs in vascular diseases, and we provide an update on the mechanisms of EPC mobilization, homing, and differentiation.

Dexamethasone downregulates calcification-inhibitor molecules and accelerates osteogenic differentiation of vascular pericytes: implications for vascular calcification.

Vascular calcification is present in many pathological conditions and is recognized as a strong predictor of future cardiovascular events. Current evidence suggests that it is a regulated process involving inducing and inhibitory molecules. Glucocorticoids have great clinical importance as antiinflammatory drugs and can act as potent inducers of osteogenic differentiation in vitro. The effect of glucocorticoids on vascular cells in vivo remains obscure. Pericytes are pluripotent cells that can differentiate into osteoblasts, and recent evidence suggests that they could participate in vascular calcification. We hypothesized that the synthetic glucocorticoid dexamethasone would enhance the rate of pericyte differentiation and mineralization in vitro with a concomitant suppression of calcification-inhibitory molecules. Three weeks of dexamethasone treatment induced a 2-fold increase in (1) alkaline phosphatase activity, (2) calcium deposition, and (3) the number of nodules formed in vitro; and a reduction in the expression of matrix Gla protein (MGP), osteopontin (OPN), and vascular calcification-associated factor (VCAF) mRNAs. The glucocorticoid receptor antagonist Org 34116 abolished dexamethasone-accelerated pericyte differentiation, nodule formation, and mineralization. Data obtained using Org 34116, the transcription inhibitor actinomycin D, and the protein synthesis inhibitor cyclohexamide suggest that MGP, OPN, and VCAF mRNA abundance are controlled at different and multiple levels by dexamethasone. This is the first report showing that dexamethasone enhances the osteogenic differentiation of pericytes and downregulates genes associated with inhibition of mineralization. Our study highlights the need for further investigation into the long-term consequences of prolonged glucocorticoid therapy on vascular calcification.

Identification and characterization of vascular calcification-associated factor, a novel gene upregulated during vascular calcification in vitro and in vivo.

OBJECTIVE: Vascular calcification, with its increasing clinical sequelae, presents an important and unresolved dilemma in cardiac and vascular practice. We aimed to identify molecules involved in this process to develop strategies for treatment or prevention. METHODS AND RESULTS: Using subtractive hybridization, a novel cDNA, designated vascular calcification-associated factor (VCAF), has been isolated from a bovine retinal pericyte cDNA library generated during the differentiation and mineralization of these cells in vitro. RNA ligase-mediated rapid amplification of cDNA ends was used to compile the 740-bp bovine cDNA sequence. Database searching reveals that VCAF has novel nucleotide/amino acid sequences. RNA analysis confirms that VCAF is upregulated in mineralized pericytes and is present in human calcified arteries but not noncalcified arteries. Protein analysis using a VCAF antibody confirms the presence of an 18-kDa protein in calcified nodules but not in confluent pericytes. Adenoviral antisense VCAF gene delivery reduces VCAF protein levels and accelerates pericyte differentiation compared with controls. CONCLUSIONS: We demonstrate the isolation of a novel gene, VCAF, which is upregulated during vascular calcification in vitro and in vivo. Antisense VCAF gene delivery accelerates pericyte differentiation, implicating a role for VCAF in this clinically significant pathological process.

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.

Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells.

AIMS: Vascular calcification (VC) is highly correlated with increased morbidity and mortality in advanced chronic kidney disease (CKD) patients. Allosteric modulation of the calcium-sensing receptor (CaR) by calcimimetics inhibits VC in animal models of advanced CKD. Here, we investigated the expression of the CaR in the vasculature and tested the ability of calcimimetics to prevent vascular smooth muscle cell (VSMC) calcification in vitro. METHODS AND RESULTS: Immunohistochemical staining demonstrated that CaR protein is present in VSMC in normal, non-calcified human arteries. In contrast, low levels of CaR immunoreactivity were detected in atherosclerotic, calcified arteries. Immunfluorescence and immunoblotting revealed that CaR protein was also expressed by human and bovine VSMC in vitro. Acute stimulation of VSMC with increased Ca2+ stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, suggesting that the VSMC CaR is functional. VSMC CaR expression decreased when these cells deposited a mineralized matrix or following 24 h incubation in mineralization medium with increased (i.e. 1.8 or 2.5 mM) Ca2+. Culturing VSMC in mineralization medium containing 1.8 and 2.5 mM Ca2+ or with the membrane-impermeant CaR agonist Gd3+ enhanced mineral deposition compared with that observed in 1.2 mM Ca2+. Over-expression of dominant-negative (R185Q) CaR enhanced, whereas the calcimimetic R-568 attenuated, VSMC mineral deposition. CONCLUSION: These results demonstrate that: (i) VSMCs express a functional CaR; (ii) a reduction in CaR expression is associated with increased mineralization in vivo and in vitro; (iii) calcimimetics decrease mineral deposition by VSMC. These data suggest that calcimimetics may inhibit the development of VC in CKD patients.