Administration of endothelial progenitor cells accelerates the resolution of arterial thrombus in mice.

BACKGROUND: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl3 lesion and in vessel re-endothelization in the mouse carotid artery. METHODS: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation. RESULTS: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro. CONCLUSIONS: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process.

Low-level laser and adipose-derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair.

OBJECTIVES: The cellular therapy using adipose-derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low-level laser (LLL), an effective photobiostimulation for the healing processes. MATERIALS AND METHODS: Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL). RESULTS: All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up-regulated Gdf5 gene expression, ASCs up-regulated the expression of Tnmd, and the association of LLL and ASCs down-regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF-α, PCNA and in the protein level of Tnmd. A higher amount of IL-10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL. CONCLUSIONS: Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.

Hyperbaric oxygen affects endothelial progenitor cells proliferation in vitro.

Hyperbaric oxygen is a clinical treatment that contributes to wound healing by increasing fibroblasts proliferation, collagen synthesis, and production of growth factors, inducing angiogenesis and inhibiting antimicrobial activity. It also has been shown that hyperbaric oxygen treatment (HBO), through the activation of nitric oxide synthase promotes an increase in the nitric oxide levels that may improve endothelial progenitor cells (EPC) mobilization from bone marrow to the peripheral blood and stimulates the vessel healing process. However, cellular mechanisms involved in cell proliferation and activation of EPC after HBO treatment remain unknown. Therefore, the present work aimed to analyze the effect of HBO on the proliferation of pre-treated bone marrow-derived EPC with TNF-alpha. Also, we investigated the expression of ICAM and eNOS by immunochemistry, the production of reactive species of oxygen and performed an in vitro wound healing. Although 1h of HBO treatment did not alter the rate of in vitro wound closure or cell proliferation, it increased eNOS expression and decreased ICAM expression and reactive oxygen species production in cells pre-treated with TNF-alpha. These results indicate that HBO can decrease the inflammatory response in endothelial cells mediated by TNF-alpha, and thus, promote vascular recovery after injury.

Combined dermatan sulfate and endothelial progenitor cell treatment: action on the initial inflammatory response after arterial injury in C57BL/6 mice.

BACKGROUND AIMS: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. METHODS: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. RESULTS: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. DISCUSSION: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.

Differentiation of C57/BL6 mice bone marrow mononuclear cells into early endothelial progenitors cells in different culture conditions.

Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.