Alteration of Notch signaling and functionality of adipose tissue derived mesenchymal stem cells in heart failure.

AIM: Circulating mesenchymal cells increase in heart failure (HF) patients and could be used therapeutically. Our aim was to investigate whether HF affects adipose tissue derived mesenchymal cell (adMSC) isolation, functional characteristics and Notch pathway. METHODS AND RESULTS: We compared 25 patients with different degrees of HF (11 NYHA classes I and II and 14 NYHA III and IV) with 10 age and gender matched controls. 100% adMSC cultures were obtained from controls, while only 72.7% and 35.7% from patients with mild or severe HF (p<0.0001). adMSC from HF patients showed higher markers of senescence (p16 positive cells: 14±2.3% in controls and 35.6±5.6% (p<0.05) and 69±14.7% (p<0.01) in mild or severe HF; γ-H2AX positive cells: 3.7±1.2%, 19.4±4.1% (p<0.05) and 23.7±3.4% (p<0.05) respectively), lower proliferation index (Ki67 positive cells: 21.5±4.9%, 13.2±2.8% and 13.7±3.2%, respectively), reduced pluripotency-associated genes (Oct4 positive cells: 86.7±4.9%, 55±12% (p<0.05) and 43.3±8.7% (p<0.05), respectively; NANOG positive cells: 89.8±3.7%, 39.6±14.4% (p<0.01) and 47±8.1%, respectively), and decreased differentiation markers (α-sarcomeric actin positive cells: 79.8±4.6%, 49±18.1% and 47±12.1% (p<0.05) and CD31-positive endothelial cells: 24.5±2.9%, 0.5±0.5% (p<0.001) and 2.3±2.3% (p<0.001), respectively). AdMSC from HF patients also showed reduced Notch transcriptional activity (lowered expression of Hey 1 and Hey 2 mRNAs). Stimulation with TNF-α of adMSC isolated from controls affected the transcription of several components of the Notch pathway (reduction of Notch 4 and Hes 1 mRNAs and increase of Notch 2 and Hey 1 mRNAs). CONCLUSIONS: In HF yield and functionality of adMSC are impaired and their Notch signaling is downregulated.

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.