A validated hypoxia-inducible factor (HIF) signature across tissue compartments predicts clinical outcome in human lung fibrosis

We previously reported that oxidative stress drives pseudohypoxic hypoxia-inducible factor (HIF) pathway activation to promote pathogenetic collagen structure-function in human lung fibrosis (Brereton et al., 2022). Here, through bioinformatic studies we investigate HIF pathway activation status in patients with idiopathic pulmonary fibrosis (IPF) and whether this has prognostic significance. Applying a well-established HIF gene expression signature, we classified publicly available datasets into HIF score-high and score-low groups across multiple tissue compartments. TheHIF scores in lung tissue, bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) were increased in IPF patients and significantly correlated with an oxidative stress signature consistent with pseudohypoxic HIF pathway activation. A high HIF score in BAL and in PBMC was a strong independent predictor of mortality in multivariate analysis. Thus, a validated HIF gene signature predicts survival across tissue compartments in IPF and merits prospective study as a non-invasive biomarker of lung fibrosis progression.

Impact of injectable chitosan cryogel microspherescaffolds on differentiation and proliferation of adiposederived mesenchymal stem cells into fat cells

Difficulty in the clinical practice of stem cell therapy is often experienced in achieving desired target tissue cell differentiation and migration of stem cells to other tissue compartments where they are destroyed or die. This study was performed to evaluate if mesenchymal stem cells (MSCs) may differentiate into desired cell types when injected after combined with an injectable cryogel scaffold and to investigate if this scaffold may help in preventing cells from passing into different tissue compartments. MSCs were obtained from fat tissue of the rabbits as autografts and nuclei and cytoplasms of these cells were labeled with BrdU and PKH26. In Group 1, only-scaffold; in Group 2, only-MSCs; and in Group 3, combined stem cell/scaffold were injected to the right malar area of the rabbits. At postoperative 3 weeks, volumes of the injected areas were calculated by computer-tomography scans and histopathological evaluation was performed. The increase in the volume of the right malar areas was more in Group 3. In histopathological evaluation, chitosan cryogel microspheres were observed microscopically within the tissue and the scaffold was only partially degraded. Normal tissue form was seen in Group 2. Cells differentiated morphologically into fat cells were detected in Groups 2 and 3. Injectable chitosan cryogel microspheres were used in vivo for the first time in this study. As it was demonstrated to be useful in carrying MSCs to the reconstructed area, help cell differentiation to desired cells and prevent migration to other tissue compartments, it may be used for reconstructive purposes in the future.

Deep Learning-Based Body Composition Analysis Predicts Outcome in Melanoma Patients Treated with Immune Checkpoint Inhibitors

Previous studies suggest an impact of body composition on outcome in melanoma patients. We aimed to determine the prognostic value of CT-based body composition assessment in patients receiving immune checkpoint inhibitor therapy for treatment of metastatic disease using a deep learning approach. One hundred seven patients with staging CT examinations prior to initiation of checkpoint inhibition between January 2013 and August 2019 were retrospectively evaluated. Using an automated deep learning-based body composition analysis pipeline, parameters for estimation of skeletal muscle mass (skeletal muscle index, SMI) and adipose tissue compartments (visceral adipose tissue index, VAI; subcutaneous adipose tissue index, SAI) were derived from staging CT. The cohort was binarized according to gender-specific median cut-off values. Patients below the median were defined as having low SMI, VAI, or SAI, respectively. The impact on outcome was assessed using the Kaplan–Meier method with log-rank tests. A multivariable logistic regression model was built to test the impact of body composition parameters on 3-year mortality. Patients with low SMI displayed significantly increased 1-year (25% versus 9%, p = 0.035), 2-year (32% versus 13%, p = 0.017), and 3-year mortality (38% versus 19%, p = 0.016). No significant differences with regard to adipose tissue compartments were observed (3-year mortality: VAI, p = 0.448; SAI, p = 0.731). On multivariable analysis, low SMI (hazard ratio (HR), 2.245; 95% confidence interval (CI), 1.005–5.017; p = 0.049), neutrophil-to-lymphocyte ratio (HR, 1.170; 95% CI, 1.076–1.273; p < 0.001), and Karnofsky index (HR, 0.965; 95% CI, 0.945–0.985; p = 0.001) remained as significant predictors of 3-year mortality. Lowered skeletal muscle index as an indicator of sarcopenia was associated with worse outcome in patients with metastatic melanoma receiving immune checkpoint inhibitor therapy.

Investigation of proteins important for microcirculation using in vivo microdialysis after glucose provocation: a proteomic study

Insulin has metabolic and vascular effects in the human body. What mechanisms that orchestrate the effects in the microcirculation, and how the responds differ in different tissues, is however not fully understood. It is therefore of interest to search for markers in microdialysate that may be related to the microcirculation. This study aims to identify proteins related to microvascular changes in different tissue compartments after glucose provocation using in vivo microdialysis. Microdialysis was conducted in three different tissue compartments (intracutaneous, subcutaneous and intravenous) from healthy subjects. Microdialysate was collected during three time periods; recovery after catheter insertion, baseline and glucose provocation, and analyzed using proteomics. Altogether, 126 proteins were detected. Multivariate data analysis showed that the differences in protein expression levels during the three time periods, including comparison before and after glucose provocation, were most pronounced in the intracutaneous and subcutaneous compartments. Four proteins with vascular effects were identified (angiotensinogen, kininogen-1, alpha-2-HS-glycoprotein and hemoglobin subunit beta), all upregulated after glucose provocation compared to baseline in all three compartments. Glucose provocation is known to cause insulin-induced vasodilation through the nitric oxide pathway, and this study indicates that this is facilitated through the interactions of the RAS (angiotensinogen) and kallikrein-kinin (kininogen-1) systems.

Mimicking extracellular matrix-mediated mechano-activation by antibodies to control signaling of the adhesion G protein-coupled receptor GPR126/ADGRG6

The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination, peripheral nerve repair and osteoblast differentiation, which renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that incorporates signals from the extracellular matrix (ECM) through binding to its N-terminal ligands collagen IV and laminin 211. Since ECM components are not suitable therapeutics alternate compounds with more favorable characteristics would be desirable that can mimic the physiologic activation pattern. Antibodies could present an apt alternative as they can specifically target the N-terminus of the receptor, reach multiple tissue compartments and have been shown to modulate GPCR activity levels.In this study, we use a monoclonal antibody targeting an N-terminal HA-epitope to induce receptor signaling, which can be enhanced through the addition of secondary antibodies. Using single cell atomic force microscopy (AFM) in combination with a fluorescent cAMP sensor, we show that antibody-mediated activation is achieved through combined pushing and pulling forces, while collagen IV and laminin 211 only mediate GPR126 activation through pushing or pulling, respectively. Thus, the antibody-mediated approach can reliably mimic the activation induced by both endogenous ligands. Our findings show that antibody-mediated activation for GPR126 is feasible and can be used for precise targeting of this receptor, thereby establishing it as a pharmaceutical target.

RNA splicing programs define tissue compartments and cell types at single cell resolution

The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes MYL6 and RPS24. These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in SAT1. Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

Targeting Drug Delivery in the Elderly: Are Nanoparticles an Option for Treating Osteoporosis?

Nanoparticles bearing specific targeting groups can, in principle, accumulate exclusively at lesion sites bearing target molecules, and release therapeutic agents there. However, practical application of targeted nanoparticles in the living organism presents challenges. In particular, intravasally applied nanoparticles encounter physical and physiological barriers located in blood vessel walls, blocking passage from the blood into tissue compartments. Whereas small molecules can pass out of the blood, nanoparticles are too large and need to utilize physiological carriers enabling passage across endothelial walls. The issues associated with crossing blood-tissue barriers have limited the usefulness of nanoparticles in clinical applications. However, nanoparticles do not encounter blood-tissue barriers if their targets are directly accessible from the blood. This review focuses on osteoporosis, a disabling and common disease for which therapeutic strategies are limited. The target sites for therapeutic agents in osteoporosis are located in bone resorption pits, and these are in immediate contact with the blood. There are specific targetable biomarkers within bone resorption pits. These present nanomedicine with the opportunity to treat a major disease by use of simple nanoparticles loaded with any of several available effective therapeutics that, at present, cannot be used due to their associated side effects.

Soft-tissue vibration and damping response to footwear changes across a wide range of anthropometrics in running

Different factors were shown to alter the vibration characteristics of soft-tissue compartments during running. Changing pre-heel strike muscle activation or changing footwear conditions represents two possibilities to influence the vibration response via frequency shift or altered damping. Associated with the study of muscle pre-tuning is the difficulty in quantifying clean experimental data for the acceleration of soft-tissue compartments and muscle activities in heterogeneous populations. The purpose of this study was to determine the vibration and pre-tuning response to footwear across a wide range of participants during running and establish and describe groups formed according to the damping coefficient. 32 subjects were used for further analysis. The subjects ran at a self-selected speed (5 min) on a treadmill in two different shoes (soft & hard), while soft-tissue accelerations and muscle activation at the gastrocnemius medialis were quantified. Damping coefficients, total muscle intensity and dominant vibration frequencies were determined. Anthropometrics and skinfold measurements of the lower limbs were obtained. According to the damping coefficient response to the footwear intervention, three groups were formed, with most runners (n = 20) showing less damping in the hard shoe. Total muscle intensity, anthropometrics, and dominant vibration frequency across footwear were not different for these three groups. Most runners (84.4%) used the strategy of adjusting the damping coefficients significantly when switching footwear. Despite damping being the preferred adjustment to changes in footwear, muscle pre-tuning might not be the only mechanism to influence damping as previously suggested. Future studies should focus on the subject-specific composition of soft-tissue compartments to elucidate their contribution to vibrations.