Micro to macro scale analysis of the intact human renal arterial tree with Synchrotron Tomography.

Background: The kidney vasculature is exquisitely structured to orchestrate renal function. Structural profiling of the vasculature in intact rodent kidneys, has provided insights into renal haemodynamics and oxygenation, but has never been extended to the human kidney beyond a few vascular generations. We hypothesised that synchrotron-based imaging of a human kidney would enable assessment of vasculature across the whole organ. Methods: An intact kidney from a 63-year-old male was scanned using hierarchical phase-contrast tomography (HiP-CT), followed by semi-automated vessel segmentation and quantitative analysis. These data were compared to published micro-CT data of whole rat kidney. Results: The intact human kidney vascular network was imaged with HiP-CT at 25 µm voxels, representing a 20-fold increase in resolution compared to clinical CT scanners. Our comparative quantitative analysis revealed the number of vessel generations, vascular asymmetry and a structural organisation optimised for minimal resistance to flow, are conserved between species, whereas the normalised radii are not. We further demonstrate regional heterogeneity in vessel geometry between renal cortex, medulla, and hilum, showing how the distance between vessels provides a structural basis for renal oxygenation and hypoxia. Conclusions: Through the application of HiP-CT, we have provided the first quantification of the human renal arterial network, with a resolution comparable to that of light microscopy yet at a scale several orders of magnitude larger than that of a renal punch biopsy. Our findings bridge anatomical scales, profiling blood vessels across the intact human kidney, with implications for renal physiology, biophysical modelling, and tissue engineering.

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

Atomic Layer Deposition of a Silver Nanolayer on Advanced Titanium Orthopedic Implants Inhibits Bacterial Colonization and Supports Vascularized de Novo Bone Ingrowth.

Joint replacement surgery is associated with significant morbidity and mortality following infection with either methicillin-resistant Staphylococcus aureus (MRSA) or Staphylococcus epidermidis. These organisms have strong biofilm-forming capability in deep wounds and on prosthetic surfaces, with 103 -104 microbes resulting in clinically significant infections. To inhibit biofilm formation, we developed 3D titanium structures using selective laser melting and then coated them with a silver nanolayer using atomic layer deposition. On bare titanium scaffolds, S. epidermidis growth was slow but on silver-coated implants there were significant further reductions in both bacterial recovery (p < 0.0001) and biofilm formation (p < 0.001). MRSA growth was similarly slow on bare titanium scaffolds and not further affected by silver coating. Ultrastructural examination and viability assays using either human bone or endothelial cells, demonstrated strong adherence and growth on titanium-only or silver-coated implants. Histological, X-ray computed microtomographic, and ultrastructural analyses revealed that silver-coated titanium scaffolds implanted into 2.5 mm defects in rat tibia promoted robust vascularization and conspicuous bone ingrowth. We conclude that nanolayer silver of titanium implants significantly reduces pathogenic biofilm formation in vitro, facilitates vascularization and osseointegration in vivo making this a promising technique for clinical orthopedic applications.

Bioactive glass foam scaffolds are remodelled by osteoclasts and support the formation of mineralized matrix and vascular networks in vitro.

Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol-gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP(+ve) ) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2 , 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 µm and 172 µm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.

Quantitation of microcomputed tomography-imaged ocular microvasculature.

PURPOSE: To quantitatively assess microvascular dimensions in the eyes of neonatal wild-type and VEGF(120)-tg mice, using a novel combination of techniques which permit three-dimensional (3D) image reconstruction. METHODS: A novel combination of techniques was developed for the accurate 3D imaging of the microvasculature and demonstrated on the hyaloid vasculature of the neonatal mouse eye. Vascular corrosion casting is used to create a stable replica of the vascular network and X-ray microcomputed tomography (muCT) to obtain the 3D images. In-house computer-aided image analysis techniques were then used to perform a quantitative morphological analysis of the images. RESULTS: With the use of these methods, differences in the numbers of vessel segments, their diameter, and volume of vessels in the vitreous compartment were quantitated in wild-type neonatal mice or littermates over-expressing a labile (nonheparin binding) isoform of vascular endothelial growth factor (VEGF(120)) from the developing lens. This methodology was instructive in demonstrating that hyaloid vascular networks in VEGFA(120) over-expressing mice have a 10-fold increase in blind-ended, a six-fold increase in connected vessel segments, in addition to a sixfold increase (0.0314 versus 0.0051 mm(3)) in total vitreous vessel volume compared with wild type. These parameters are not readily quantified via histological, ultrastructural, or stereological analysis. CONCLUSION: The combination of techniques described here provides the first 3D quantitative characterization of vasculature in an organ system; i.e., the neonatal murine intra-ocular vasculature in both wild-type mice and a transgenic model of lens-specific over-expression of VEGF.

Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies.

Control genes, commonly defined as genes that are ubiquitously expressed at stable levels in different biological contexts, have been used to standardize quantitative expression studies for more than 25 yr. We analyzed a group of large mammalian microarray datasets including the NCI60 cancer cell line panel, a leukemia tumor panel, and a phorbol ester induction time course as well as human and mouse tissue panels. Twelve housekeeping genes commonly used as controls in classical expression studies (including GAPD, ACTB, B2M, TUBA, G6PD, LDHA, and HPRT) show considerable variability of expression both within and across microarray datasets. Although we can identify genes with lower variability within individual datasets by heuristic filtering, such genes invariably show different expression levels when compared across other microarray datasets. We confirm these results with an analysis of variance in a controlled mouse dataset, showing the extent of variability in gene expression across tissues. The results show the problems inherent in the classical use of control genes in estimating gene expression levels in different mammalian cell contexts, and highlight the importance of controlled study design in the construction of microarray experiments.

Expression profiles of 290 ESTs mapped to chromosome 3 in human epithelial ovarian cancer cell lines using DNA expression oligonucleotide microarrays.

We have investigated previously the utility of oligonucleotide expression microarray technology in an analysis of four spontaneously transformed epithelial ovarian cancer (EOC) cell lines, TOV-21G, TOV-81D, OV-90, and TOV-112D. Here, we examine the expression of 290 expressed sequence tags (ESTs) that map to human chromosome 3 in a primary culture derived from normal ovarian surface epithelium (NOSE), NOV-31, and the four spontaneously transformed EOC cell lines. One of these cell lines, OV-90, harbors a deletion of an entire chromosome 3p arm. Whereas the most aggressive cell lines (OV-90, TOV-112D, and TOV-21G) exhibited the highest levels of expression, assessed by the mean of expression values of all ESTs, OV-90 showed the lowest mean of expression of ESTs that map to the 3p arm in comparison with TOV-112D and TOV-21G. This difference in expression profile of 3p ESTs in OV-90 is also reflected in the ratio of expression of ESTs on 3p versus the 3q arm and in that the expression values of ESTs that map to 3p were more often lower than higher in OV-90 in two-way comparisons with NOV-31, TOV-21G, and TOV-112D. The loss of a 3p arm does not affect the pattern of differential expression in analyses based on the range of numeric expression values of each EST or fold differences in expression for each EST in comparison with NOV-31. However, 25 differentially expressed ESTs were identified on the basis of threefold differences in expression values between NOV-31 and any EOC cell line; and six of these ESTs were differentially expressed uniquely in OV-90. The investigation of these ESTs could facilitate the identification of novel chromosome 3 genes implicated in ovarian tumorigenesis.