The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

Dose response of endotoxin on hepatocyte and muscle mitochondrial respiration in vitro.

INTRODUCTION Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. METHODS Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1-100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. RESULTS In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). CONCLUSION LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.