The Cilialyzer - A freely available open-source software for the analysis of mucociliary activity in respiratory cells.

BACKGROUND AND OBJECTIVE: Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and associated airway disease. As there is currently no single diagnostic gold standard test, PCD is diagnosed by a combination of several methods comprising genetic testing and the examination of the ciliary structure and function. Among the approved diagnostic methods, only high-speed video microscopy (HSVM) allows to directly observe the ciliary motion and therefore, to directly assess ciliary function. In the present work, we present our recently developed freely available open-source software - termed "Cilialyzer", which has been specifically designed to support and facilitate the analysis of the mucociliary activity in respiratory epithelial cells captured by high-speed video microscopy. METHODS: In its current state, the Cilialyzer software enables clinical PCD analysts to load, preprocess and replay recorded image sequences as well as videos with a feature-rich replaying module facilitating the commonly performed qualitative visual assessment of ciliary function (including the assessment of the ciliary beat pattern). The image processing methods made accessible through an intuitive user interface allow clinical specialists to comfortably compute the ciliary beating frequency (CBF), the activity map and the "frequency correlation length" - an observable getting newly introduced. Furthermore, the Cilialyzer contains a simple-to-use particle tracking interface to determine the mucociliary transport speed. RESULTS: Cilialyzer is fully written in the Python programming language and freely available under the terms of the MIT license. The proper functioning of the computational analysis methods constituting the Cilialyzer software is demonstrated by using simulated and representative sample data from clinical practice. Additionally, the software was used to analyze high-speed videos showing samples obtained from healthy controls and genetically confirmed PCD cases (DNAI1 and DNAH11 mutations) to show its clinical applicability. CONCLUSIONS: Cilialyzer serves as a useful clinical tool for PCD analysts and provides new quantitative information awaiting to be clinically evaluated using cohorts of PCD. As Cilialyzer is freely available under the terms of a permissive open-source license, it serves as a ground frame for further development of computational methods aiming at the quantification and automation of the analysis of mucociliary activity captured by HSVM.

In Vitro Effect of Combined Hypertonic Saline and Salbutamol on Ciliary Beating Frequency and Mucociliary Transport in Human Nasal Epithelial Cells of Healthy Volunteers and Patients with Cystic Fibrosis.

Background: Inhalation of hypertonic saline (HS) is standard of care in patients with cystic fibrosis (CF). However, it is unclear if adding salbutamol has-besides bronchodilation-further benefits, for example, on the mucociliary clearance. We assessed this in vitro by measuring the ciliary beating frequency (CBF) and the mucociliary transport rate (MCT) in nasal epithelial cells (NECs) of healthy volunteers and patients with CF. Aims: To investigate the effect of HS, salbutamol, and its combination on (muco)ciliary activity of NECs in vitro, and to assess potential differences between healthy controls and patients with CF. Methods: NECs obtained from 10 healthy volunteers and 5 patients with CF were differentiated at the air-liquid interface and aerosolized with 0.9% isotonic saline ([IS] control), 6% HS, 0.06% salbutamol, or combined HS and salbutamol. CBF and MCT were monitored over 48-72 hours. Results: In NECs of healthy controls, the absolute CBF increase was comparable for all substances, but CBF dynamics were different: HS increased CBF slowly and its effect lasted for an extended period, salbutamol and IS increased CBF rapidly and the effect subsided similarly fast, and HS and salbutamol resulted in a rapid and long-lasting CBF increase. Results for CF cells were comparable, but less pronounced. Similar to CBF, MCT increased after the application of all the tested substances. Conclusion: CBF and MCT of NECs of healthy participants and CBF of patients with CF increased upon treatment with aerosolized IS, HS, salbutamol, or HS and salbutamol, showing a relevant effect for all tested substances. The difference in the CBF dynamics can be explained by the fact that the properties of the mucus are changed differently by different saline concentrations.

Impact of cerebral hypoperfusion-reperfusion on optic nerve integrity and visual function in the DBA/2J mouse model of glaucoma.

OBJECTIVE: One of the most important risk factors for developing a glaucomatous optic neuropathy is elevated intraocular pressure. Moreover, mechanisms such as altered perfusion have been postulated to injure the optical path. In a mouse model, we compare first negative effects of cerebral perfusion/reperfusion on the optic nerve structure versus alterations by elevated intraocular pressure. Second, we compare the alterations by isolated hypoperfusion-reperfusion and isolated intraocular pressure to the combination of both. METHODS AND ANALYSIS: Mice were divided in four groups: (1) controls; (2) perfusion altered mice that underwent transient bi-common carotid artery occlusion (BCCAO) for 40 min; (3) glaucoma group (DBA/2J mice); (4) combined glaucoma and altered perfusion (DBA/2J mice with transient BCCAO). Optic nerve sections were stereologically examined 10-12 weeks after intervention. RESULTS: All experimental groups showed a decreased total axon number per optic nerve compared with controls. In DBA/2J and combined DBA/2J & BCCAO mice the significant decrease was roughly 50%, while BCCAO leaded to a 23% reduction of axon number, however reaching significance only in the direct t-test. The difference in axon number between BCCAO and both DBA/2J mice was almost 30%, lacking statistical significance due to a remarkably high variation in both DBA/2J groups. CONCLUSION: Elevated intraocular pressure in the DBA/2J mouse model of glaucoma leads to a much more pronounced optic nerve atrophy compared with transient forebrain hypoperfusion and reperfusion by BCCAO. A supposed worsening effect of an altered perfusion added to the pressure-related damage could not be detected.

Pulmonary acini exhibit complex changes during postnatal rat lung development.

Pulmonary acini represent the functional gas-exchanging units of the lung. Due to technical limitations, individual acini cannot be identified on microscopic lung sections. To overcome these limitations, we imaged the right lower lobes of instillation-fixed rat lungs from postnatal days P4, P10, P21, and P60 at the TOMCAT beamline of the Swiss Light Source synchrotron facility at a voxel size of 1.48 µm. Individual acini were segmented from the three-dimensional data by closing the airways at the transition from conducting to gas exchanging airways. For a subset of acini (N = 268), we followed the acinar development by stereologically assessing their volume and their number of alveoli. We found that the mean volume of the acini increases 23 times during the observed time-frame. The coefficients of variation dropped from 1.26 to 0.49 and the difference between the mean volumes of the fraction of the 20% smallest to the 20% largest acini decreased from a factor of 27.26 (day 4) to a factor of 4.07 (day 60), i.e. shows a smaller dispersion at later time points. The acinar volumes show a large variation early in lung development and homogenize during maturation of the lung by reducing their size distribution by a factor of 7 until adulthood. The homogenization of the acinar sizes hints at an optimization of the gas-exchange region in the lungs of adult animals and that acini of different size are not evenly distributed in the lungs. This likely leads to more homogeneous ventilation at later stages in lung development.

Diagnosis of primary ciliary dyskinesia: discrepancy according to different algorithms.

BACKGROUND: Diagnosis of primary ciliary dyskinesia (PCD) is challenging since there is no gold standard test. The European Respiratory (ERS) and American Thoracic (ATS) Societies developed evidence-based diagnostic guidelines with considerable differences. OBJECTIVE: We aimed to compare the algorithms published by the ERS and the ATS with each other and with our own PCD-UNIBE algorithm in a clinical setting. Our algorithm is similar to the ERS algorithm with additional immunofluorescence staining. Agreement (Cohen's κ) and concordance between the three algorithms were assessed in patients with suspicion of PCD referred to our diagnostic centre. RESULTS: In 46 out of 54 patients (85%) the final diagnosis was concordant between all three algorithms (30 PCD negative, 16 PCD positive). In eight patients (15%) PCD diagnosis differed between the algorithms. Five patients (9%) were diagnosed as PCD only by the ATS, one (2%) only by the ERS and PCD-UNIBE, one (2%) only by the ATS and PCD-UNIBE, and one (2%) only by the PCD-UNIBE algorithm. Agreement was substantial between the ERS and the ATS (κ=0.72, 95% CI 0.53-0.92) and the ATS and the PCD-UNIBE (κ=0.73, 95% CI 0.53-0.92) and almost perfect between the ERS and the PCD-UNIBE algorithms (κ=0.92, 95% CI 0.80-1.00). CONCLUSION: The different diagnostic algorithms lead to a contradictory diagnosis in a considerable proportion of patients. Thus, an updated, internationally harmonised and standardised PCD diagnostic algorithm is needed to improve diagnostics for these discordant cases.

A Comprehensive Approach for the Diagnosis of Primary Ciliary Dyskinesia-Experiences from the First 100 Patients of the PCD-UNIBE Diagnostic Center.

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.

Multi-scale alignment of respiratory cilia and its relation to mucociliary function.

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance - the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness.

EpCAM+CD73+ mark epithelial progenitor cells in postnatal human lung and are associated with pathogenesis of pulmonary disease including lung adenocarcinoma.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5'-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin ß4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air-liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.

Structural Microangiopathies in Skeletal Muscle Related to Systemic Vascular Pathologies in Humans.

It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication - peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7-39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies.

Multi-walled carbon nanotubes activate and shift polarization of pulmonary macrophages and dendritic cells in an in vivo model of chronic obstructive lung disease.

With substantial progress of nanotechnology, there is rising concern about possible adverse health effects related to inhalation of nanomaterials, such as multi-walled carbon nanotubes (MWCNT). In particular, individuals with chronic respiratory disorders, such as chronic obstructive pulmonary disease (COPD), may potentially be more susceptible to adverse health effects related to inhaled MWCNT. Hazard assessment of such inhaled nanomaterials therefore requires timely clarification. This was assessed in this study using a mouse model of COPD by exposing animals to 0.08 µg/cm2 of MWCNT administered by intratracheal instillation. Treatment with MWCNT induced an accumulation of alveolar macrophages (AMφ) in bronchoalveolar lavage fluid (BALF) in COPD mice that increased from 24 h to 7 d. In COPD mice, MWCNT induced a dynamic shift in macrophage polarization as measured by expression of CD38 and CD206, and increased AMφ and lung parenchyma macrophage (LPMΦ) activation with upregulation of co-stimulatory markers CD40 and CD80. Moreover, MWCNT treatment increased the frequencies of pulmonary dendritic cells (DC), leading to an expansion of the CD11b+CD103- DC subset. Although MWCNT did not trigger lung functional or structural changes, they induced an increased expression of the muc5AC transcript in mice with COPD. Our data provide initial evidence that inhaled MWCNT affect the pulmonary mucosal immune system by altering the numbers, phenotype, and activation status of antigen-presenting cell populations. Extrapolating these in vivo mouse findings to human pulmonary MWCNT exposure, caution is warranted in limiting exposure when handling inhalable nanofibers.

Acute effects of multi-walled carbon nanotubes on primary bronchial epithelial cells from COPD patients.

The risks of occupational exposure during handling of multi-walled carbon nanotubes (MWCNTs) have received limited attention to date, in particular for potentially susceptible individuals with highly prevalent chronic obstructive pulmonary disease (COPD). In this in vitro study, we simulated acute inhalation of MWCNTs employing an air-liquid interface cell exposure (ALICE) system: primary human bronchial epithelial cells from COPD patients and healthy donors (controls), cultured at the air-liquid interface (ALI) were exposed to MWCNTs. To study acute health effects on the respiratory epithelium, two different concentrations (0.16; 0.34 µg/cm2) of MWCNTs were aerosolized onto cell cultures followed by analysis after 24 h. Following MWCNT exposure, epithelial integrity and differentiation remained intact. Electron microscopy analyses identified MWCNTs both extra- and intracellular within vesicles of mucus producing cells. In both COPD and healthy control cultures, MWCNTs neither caused increased release of lactate dehydrogenase (LDH), nor alterations in inflammatory responses, as measured by RNA expression and protein secretion of the cytokines IL-6, IL-8, CXCL10, IL-1ß and TGF-ß and oxidative stress markers HMOX-1 and SOD-2. No short-term alteration of epithelial cell function, as determined by ciliary beating frequency (CBF), occurred in any of the conditions tested. In conclusion, the present study provided a reliable and realistic in vitro acute-exposure model of the respiratory tract, responsive to positive controls such as Dörentruper Quartz (DQ12) and asbestos. Acute exposure to MWCNTs did not affect epithelial integrity, nor induce increased cell death, apoptosis or inflammatory changes.

The influence of age on valve disease in patients with varicose veins analysed by transmission electron microscopy and stereology.

BACKGROUND: The aim of this study was to investigate the influence of age on the ultrastructure of venous valve morphology in patients with C2 classified chronic venous disorders according to the CEAP classification. PATIENTS AND METHODS: The study population consisted of 16 consecutive patients with varicose veins (C2). The mean age was 49.8 years (30-66). The (pre-) terminal valve including the vessel wall was harvested within the proximal 2 centimetres of the great saphenous vein. The mean thickness (volume-to-surface ratio = V/S ratio) of elastin, collagen, endothelium and of the entire valve was determined. A blinded morphologist performed the examination by transmission electron microscopy and stereology. Analyses by Pearson's product moment correlation, Kendall's tau and Spearman's rank correlation were performed to investigate whether there is a correlation between age and the ultrastructural morphology. RESULTS: Stereological analysis of the valves demonstrated a mean V/S ratio (signifying a thickness estimation) for elastin of 0.87 µm3/µm2, for collagen of 18.0 µm3/µm2, for endothelium of 0.65 µm3/µm2, and for the entire valve of 25.2 µm³/µm². Statistical analyses showed no statistically significant correlation between age and the ultrastructural morphology in this patient group. CONCLUSIONS: The ultrastructural morphology of the venous valves in chronic venous disorders may not depend on age in patients presenting with C2 disease. This conclusion may or may not apply to all C classes as we investigated a homogenous group of patients with C2 limbs.

Increased capillary tortuosity and pericapillary basement membrane thinning in skeletal muscle of mice undergoing running wheel training.

To work out which microvascular remodeling processes occur in murine skeletal muscle during endurance exercise, we subjected C57BL/6 mice to voluntary running wheel training for 1 week (1 wk-t) or 6 weeks (6 wks-t). By means of morphometry, the capillarity as well as the compartmental and sub-compartmental structure of the capillaries were quantitatively described at the light microscopy level and at the electron microscopy level, respectively, in the plantaris (PLNT) muscle of the exercising mice in comparison to untrained littermates. In the early phase of the training (1 wk-t), angiogenesis [32% higher capillary/fiber (C/F) ratio; P<0.05] in PLNT muscle was accompanied by a tendency for capillary lumen enlargement (30%; P=0.06) and a reduction of the pericapillary basement membrane thickness [(CBMT) 12.7%; P=0.09] as well as a 21% shortening of intraluminal protrusion length (P<0.05), all compared with controls. After long-term training (6 wks-t), when the mice reached a steady state in running activity, additional angiogenesis (C/F ratio: 76%; P<0.05) and a 16.3% increase in capillary tortuosity (P<0.05) were established, accompanied by reversal of the lumen expansion (23%; P>0.05), further reduction of the CBMT (16.5%; P<0.05) and additional shortening of the intraluminal protrusion length (23%; P<0.05), all compared with controls. Other structural indicators, such as capillary profile sizes, profile area densities, perimeters of the capillary compartments and concentrations of endothelium-pericyte peg-socket junctions, were not significantly different between the mouse groups. Besides angiogenesis, increase of capillary tortuosity and reduction of CBMT represent the most striking microvascular remodeling processes in skeletal muscle of mice that undergo running wheel training.

Cutting-edge microangio-CT: new dimensions in vascular imaging and kidney morphometry.

In the last decades, the contrast-enhanced micro-computed tomography (micro-CT) imaging of a whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop micro-CT scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature and glomeruli number. This method is based on µAngiofil, a new polymerizing contrast agent with homogenous X-ray absorption, which provides continuous filling of the complete vasculature and enables correlative imaging approaches. For rapid and reliable kidney morphometry, the microangio-CT (µaCT) data sets from glial cell line-derived neurotrophic factor (GDNF)+/- mice and their wild-type littermates were used. The results were obtained much faster compared with the current gold standard, histology-based stereology, and without processing artifacts. The histology-based morphometry was done afterward on the same kidneys. Both approaches revealed that the GDNF+/- male mice had about 40% fewer glomeruli. Furthermore, our approach allows for the definition of sites of interest for further histological investigation, i.e., correlative morphology. The polymerized µAngiofil stays in perfused vessels and is autofluorescent, which is what greatly facilitates the matching of histological sections with µaCT data. The presented approach is a time-efficient, reliable, qualitative, and quantitative methodology. Besides glomerular morphometry, the µaCT data can be used for qualitative and quantitative analysis of the kidney vasculature and correlative morphology.

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling.

BACKGROUND: In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients. METHODS: Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay. RESULTS: Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight. CONCLUSION: In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle-Specific VEGF Expression.

Vascular endothelial growth factor-A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle-specific VEGF knockout (mVEGF-KO, n = 7) mice and their wild-type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: -63.5%; soleus: -54.8%; P ≤ 0.05) in mVEGF-KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv-values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF-KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse-strains: Vv (-17.4%), absolute area size (-19.1%) and thickness (-19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF-KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF-KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239-2249, 2017. © 2017 Wiley Periodicals, Inc.

Capillary ultrastructure and mitochondrial volume density in skeletal muscle in relation to reduced exercise capacity of patients with intermittent claudication.

Intermittent claudication (IC) is the most commonly reported symptom of peripheral arterial disease (PAD). Impaired limb blood flow is a major casual factor of lower exercise tolerance in PAD but cannot entirely explain it. We hypothesized that IC is associated with structural changes of the capillary-mitochondria interface that could contribute to the reduction of exercise tolerance in IC patients. Capillary and mitochondrial morphometry were performed after light and transmission electron microscopy using vastus lateralis muscle biopsies of 14 IC patients and 10 age-matched controls, and peak power output (PPO) was determined for all participants using an incremental single-leg knee-extension protocol. Capillary density was lower (411 ± 90 mm(-2) vs. 506 ± 95 mm(-2); P ≤ 0.05) in the biopsies of the IC patients than in those of the controls. The basement membrane (BM) around capillaries was thicker (543 ± 82 nm vs. 423 ± 97 nm; P ≤ 0.01) and the volume density of mitochondria was lower (3.51 ± 0.56% vs. 4.60 ± 0.74%; P ≤ 0.01) in the IC patients than the controls. In the IC patients, a higher proportion of capillaries appeared with collapsed slit-like lumen and/or swollen endothelium. PPO was lower (18.5 ± 9.9 W vs. 33.5 ± 9.4 W; P ≤ 0.01) in the IC patients than the controls. We suggest that several structural alterations in skeletal muscle, either collectively or separately, contribute to the reduction of exercise tolerance in IC patients.

Zebrafish Caudal Fin Angiogenesis Assay-Advanced Quantitative Assessment Including 3-Way Correlative Microscopy.

BACKGROUND: Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics. OBJECTIVE: To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way. APPROACH & RESULTS: Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including "graph energy" and "distance to farthest node". The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level. CONCLUSIONS: The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.

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.