nNOS Increases Fiber Type-Specific Angiogenesis in Skeletal Muscle of Mice in Response to Endurance Exercise.

We studied the relationship between neuronal NO synthase (nNOS) expression and capillarity in the tibialis anterior (TA) muscle of mice subjected to treadmill training. The mRNA (+131%) and protein (+63%) levels of nNOS were higher (p ≤ 0.05) in the TA muscle of C57BL/6 mice undergoing treadmill training for 28 days than in those of littermates remaining sedentary, indicating an up-regulation of nNOS by endurance exercise. Both TA muscles of 16 C57BL/6 mice were subjected to gene electroporation with either the pIRES2-ZsGreen1 plasmid (control plasmid) or the pIRES2-ZsGreen1-nNOS gene-inserted plasmid (nNOS plasmid). Subsequently, one group of mice (n = 8) underwent treadmill training for seven days, while the second group of mice (n = 8) remained sedentary. At study end, 12-18% of TA muscle fibers expressed the fluorescent reporter gene ZsGreen1. Immunofluorescence for nNOS was 23% higher (p ≤ 0.05) in ZsGreen1-positive fibers than ZsGreen1-negative fibers from the nNOS-transfected TA muscle of mice subjected to treadmill training. Capillary contacts around myosin heavy-chain (MHC)-IIb immunoreactive fibers (14.2%; p ≤ 0.05) were only higher in ZsGreen1-positive fibers than ZsGreen1-negative fibers in the nNOS-plasmid-transfected TA muscles of trained mice. Our observations are in line with an angiogenic effect of quantitative increases in nNOS expression, specifically in type-IIb muscle fibers after treadmill training.

Molecular Assessment of Staphylococcus Aureus Strains in STAT3 Hyper-IgE Syndrome Patients.

Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI) caused by monogenic defects such as in the gene STAT3 (STAT3-HIES). Patients suffering from HIES show an increased susceptibility to Staphylococcus aureus (S. aureus) including skin abscesses and pulmonary infections. To assess if the underlying immune defect of STAT3-HIES patients influences the resistance patterns, pathogenicity factors or strain types of S. aureus. We characterized eleven S. aureus strains isolated from STAT3-HIES patients (n = 4) by whole genome sequencing (WGS) to determine presence of resistance and virulence genes. Additionally, we used multi-locus sequence typing (MLST) and protein A (spa) typing to classify these isolates. Bacterial isolates collected from this cohort of STAT3-HIES patients were identified as common spa types in Germany. Only one of the isolates was classified as methicillin-resistant S. aureus (MRSA). For one STAT3 patient WGS illustrated that infection and colonization occurred with different S. aureus isolates rather than one particular clone. The identified S. aureus carriage profile on a molecular level suggests that S. aureus strain type in STAT3-HIES patients is determined by local epidemiology rather than the underlying immune defect highlighting the importance of microbiological assessment prior to antibiotic treatment.

Reduced Immunoglobulin (Ig) G Response to Staphylococcus aureus in STAT3 Hyper-IgE Syndrome.

STAT3 hyper-IgE syndrome (STAT3-HIES) patients presented with significantly lower Staphylococcus aureus-specific serum IgG compared to cystic fibrosis patients despite recurrent S. aureus infections. Immunoglobulin replacement therapy increased S. aureus-specific IgG in STAT3-HIES patients and attenuated the clinical course of disease suggesting a role of humoral immunity in S. aureus clearance.

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 angiotensin converting enzyme insertion/deletion polymorphism alters the response of muscle energy supply lines to exercise.

The presence of a silencing sequence (the I-allele) in the gene for the upstream regulator of blood flow, angiotensin I-converting enzyme (ACE), is associated with superior endurance performance and its trainability. We tested in a retrospective study with 36 Caucasian men of Swiss descent whether carriers of the ACE I-allele demonstrate a modified adaptive response of energy supply lines in knee extensor muscle, and aerobic fitness, to endurance training based on 6 weeks of supervised bicycle exercise or 6 months of self-regulated running (p value

Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle.

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

The beta-isoform of neuronal nitric oxide synthase (nNOS) lacking the PDZ domain is localized at the sarcolemma.

In skeletal muscles, the expression of neuronal NO synthase (nNOS) isoforms is uncharacterized at the protein level. We therefore conducted epitope mapping with anti-peptide-antibodies. Antibodies specific for the nNOS N-terminus recognized the 160-kDa alpha-isoform. In contrast, antibodies against the middle portion or the C-terminus of nNOS bound additionally to the truncated 140-kDa beta-isoform which lacks the PDZ-domain present in the alpha-isoform. All nNOS immunohistochemical reactivity was confined to the sarcolemma. Consistently, immunoblotting disclosed both nNOS-isoforms to be co-enriched in the membrane-associated fractions. The beta-isoform was co-immunoprecipitated with alpha-isoform antibodies in muscle extracts indicating an association of both nNOS-isoforms to direct the beta-variant to the sarcolemma.