An inflammatory process may be involved in nitric oxide production in skeletal muscle of type 2 diabetic patients. Nitric oxide generation in skeletal muscle was assessed in 14 non-complicated type 2 diabetic patients and in 12 healthy subjects. In samples of quadriceps femoris muscle, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrite, nitrate and nitrotyrosine were determined. The macrophage-specific antigen CD163, the T-cell membrane factor CD154 and tumour necrosis factor-alpha (TNF-alpha) were also assayed. In six patients, ultrastructural analysis of muscle was performed. Nitrites and nitrates were increased in patients as compared to controls (22.7+/-4.5 and 32.7+/-7.0 vs 16.0+/-2.9 and 22.8+/-4.0 micromol/mg protein; P<0.001, Mann-Whitney U test). Endothelial NOS was similar in diabetic and control subjects (36.4+/-13.8 vs 36.3+/-6.8 ng/mg protein), contrasting with the significant increase of iNOS recorded in patients (34.3+/-13.0 vs 8.5+/-2.8 ng/mg protein, P<0.00002). Nitrotyrosine levels were higher in the patient than in the control group (42.1+/-24.4 vs 10.3+/-2.5 ng/mg protein, P<0.00002), as were CD163 (10-fold) and TNF-alpha (fourfold) levels. Furthermore, CD154 levels were detectable only in the patient samples (10.2+/-5.3 ng/mg protein). By multiple-regression analysis, changes in glycated haemoglobin values could predict 96% variation in nitrotyrosine. Macrophages were present in all muscle samples analysed by electromicroscopy. The increased levels of CD163, CD154 and TNF-alpha indicate that an inflammatory process occurs in skeletal muscle of type 2 diabetic patients. This may contribute to iNOS induction, muscle damage and insulin resistance.

Aim The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high‐density lipoprotein (HDL), to endothelium‐dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto‐F1‐ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)‐sensitive P2Y receptors (P2Y ADP receptors). Ecto‐F1‐ATPase is involved in APOA1–mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid‐free APOA1 and the involvement of ecto‐F1‐ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. Method Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. Results Physiological concentrations of lipid‐free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto‐F1‐ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto‐F1‐ATPase and P2Y1 receptor. Conclusions Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto‐F1‐ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

BackgroundMesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo.MethodsMesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).ResultshWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.ConclusionThese results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

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