Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is α-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Intravenous administration of allogeneic Wharton jelly-derived mesenchymal stem cells for treatment of dogs with congestive heart failure secondary to myxomatous mitral valve disease.

OBJECTIVE: To evaluate whether mesenchymal stem cells (MSCs) can be safely administered IV to dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve disease (MMVD) to improve cardiac function and prolong survival time. ANIMALS: 10 client-owned dogs with CHF secondary to MMVD. PROCEDURES: Dogs with an initial episode of CHF secondary to MMVD were enrolled in a double-blind, placebo-controlled clinical trial. Five dogs in the MSC group received allogeneic Wharton jelly-derived MSCs (2 × 106 cells/kg, IV), and 5 dogs in the placebo group received a 1% solution of autologous serum (IV) for 3 injections 3 weeks apart. Cell-release criteria included trilineage differentiation, expression of CD44 and CD90 and not CD34 and major histocompatability complex class II, normal karyotype, and absence of contamination by pathogenic microorganisms. Patients were followed for 6 months or until death or euthanasia. Echocardiographic data, ECG findings, serum cardiac biomarker concentrations, CBC, and serum biochemical analysis results were obtained prior to and 4 hours after the first injection and every 3 months after the final injection. RESULTS: Lymphocyte and eosinophil counts decreased significantly 4 hours after injection, and monocytes decreased significantly only in dogs that received an MSC injection. No significant differences were seen in the echocardiographic variables, ECG results, serum cardiac biomarker concentrations, survival time, and time to first diuretic drug dosage escalation between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: This study showed that MSCs can be easily collected from canine Wharton jelly as an allogeneic source of MSCs and can be safely delivered IV to dogs with CHF secondary to MMVD.

Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis.

Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.

Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells Suppress CD4 Expressing T Cells Through Transforming Growth Factor Beta and Adenosine Signaling in a Canine Model.

Mesenchymal stem cells (MSCs) are widely investigated as potential therapeutic agents due to their potent immunomodulatory capacity. Although specific mechanisms by which MSC acts on immune cells are emerging, many questions remain, including the potential of extracellular vesicles (EVs) to mediate biological activities. Canine MSCs are of interest for both veterinary and comparative models of disease and have been shown to suppress CD4pos T cell proliferation. The aim of this study was to determine whether EV isolated from canine Wharton's jelly-derived MSC (WJ-MSC EV) suppresses CD4pos T cell proliferation using biochemical mechanisms previously ascribed to soluble mediators [transforming growth factor beta (TGF-ß) and adenosine]. WJ-MSC EV exhibited mode of 125 nm diameter, low buoyant density (1.1 g/mL), and expression of EV proteins Alix and TSG101. Functionally, EVs inhibited CD4pos T cell proliferation in a dose-dependent manner, which was absent in EV-depleted samples and EVs from non-MSC fibroblasts. EV suppression of CD4pos T cell proliferation was inhibited by a TGF-ßRI antagonist, neutralizing antibodies to TGF-ß, or A2A adenosine receptor blockade. TGF-ß was present on EVs as latent complexes most likely tethered to EV membrane by betaglycan. These data demonstrate that canine WJ-MSC EV utilizes TGF-ß and adenosine signaling to suppress proliferation of CD4pos T cell and will enable further investigation into mechanisms of immune cell modulation, as well as refinement of WJ-MSC and their EVs for therapeutic application.

Practical Methods for Assessing Emphysema Severity Based on Estimation of Linear Mean Intercept (Lm) in the Context of Animal Models of Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency is typified by panacinar emphysema in humans. Whilst animal models of (α1A-TD) that more accurately reflect the histology and molecular pathology of α1A-TD are in development, it is timely to discuss methods to assess emphysema severity. Several methods exist to quantify emphysema from histologic sections, including linear mean intercept (Lm), equivalent diameters (D) or their statistical derivatives (D2), and more recently probability models of D2 ("severity index"). Given proper attention to lung inflation, reference volume, and random sampling, Lm determined by intersect point counting provides a robust analytical tool to quantify emphysema severity. Details of lung preparation, processing for random sampling, and batch processing of prescreened images are provided herein.

Circulating exosome microRNA associated with heart failure secondary to myxomatous mitral valve disease in a naturally occurring canine model.

Myxomatous mitral valve disease (MMVD) is functionally and histologically identical to mitral valve prolapse (MVP) in humans. Currently, there are no medical treatments that can delay the progression of this valvular disease or associated cardiac remodelling. Therefore, there is a need to understand the molecular pathology associated with MMVD and MVP better, and thus identify potential therapeutic targets. Circulating exosomes contain small RNA, including miRNA, which reflect cell physiology and pathology. This study explored the association between circulating exosomal miRNA (ex-miRNA) content and MMVD, heart failure due to MMVD (MMVD-CHF) and ageing, which is strongly associated with MMVD. Ex-miRNA was isolated from old normal/healthy dogs (n = 6), young normal dogs (n = 7), dogs with MMVD (n = 7) and dogs with MMVD-CHF (n = 7). Separately, total plasma miRNA was isolated from normal dogs (n = 8), dogs with MMVD (n = 8) and dogs with MMVD-CHF (n = 11). Using reverse transcription quantitative polymerase chain reaction, exosomal miR-181c (p = 0.003) and miR-495 (p = 0.0001) significantly increased in dogs with MMVD-CHF compared to the other three groups. Exosomal miR-9 (p = 0.002) increased in dogs with MMVD and MMVD-CHF compared to age-matched (old) normal dogs. Exosomal miR-599 (p = 0.002) decreased in dogs with MMVD compared to old normal dogs. In total plasma, 58 miRNA were deemed significantly different (p < 0.04) between normal dogs, dogs with MMVD and dogs with MMVD-CHF. However, in contrast to ex-miRNA, none of the miRNA in total plasma remained statistically significant if the false discovery rate was <15%. Changes in ex-miRNA are observed in dogs as they age (miR-9, miR-495 and miR-599), develop MMVD (miR-9 and miR-599) and progress from MMVD to CHF (miR-181c and miR-495). Ex-miRNA expression-level changes appear to be more specific to disease states than total plasma miRNA. RESPONSIBLE EDITOR Elena Aikawa, Harvard Medical School, USA.

Age dependence of lung mesenchymal stromal cell dynamics following pneumonectomy.

Aging is a critical determinant of regenerative capacity in many organ systems, but it remains unresolved in the lung. This study examines murine lung cell dynamics during age-dependent lung regeneration. Proliferation of lung progenitor cells (EpCAM(neg)/Sca-1(high) lung mesenchymal stromal cells - LMSCs, EpCAM(pos)/Sca-1(low) epithelial progenitor cells, proSP-C(pos) alveolar type II epithelial cells - AECII, and CD31(pos) - endothelial cells) was tracked to day 3 or 7 after pneumonectomy (PNX) or SHAM surgery in 3, 9, and 17 month mice. In 3 month mice, post-PNX LMSC proliferation peaked early (3 days), with 50%-80% more BrdU-positive cells than the other cell types, which peaked later (4-7 days). In older mice (9 and 17 month), abundance and post-PNX proliferation of LMSCs at day 3 were reduced (40%-80%). In both young and old mice, LMSCs were isolated and compared phenotypically with whole lung non-LMSCs. Donor age had no qualitative effect on the phenotype (LMSC vs. non-LMSC), with increased expression of CD90/Thy1, CD105/Eng, CD106/Vcam, CD146/Mcam, and Pdgfrα, and up-regulation of mRNA encoding Fap, Eln, Col1a1, Col3a1, Aldh1a3, Arhgef25, Dner, Fgfr1, and Midkine. However, compared with LMSCs isolated from young mice, LMSCs from older mice exhibited reduced mRNA expression of retinoic acid (Aldh1a3, Rbp4), Fgf/Wnt (Fgfr1, Sfrp1, Wnt2, and Ctnnb1), and elastogenesis (Col1a1, Eln, Fbn1, and Sdc2) pathway genes. Isolated LMSCs from older mice also demonstrated lower colony-forming units (-67%), growth potential (-60% by day 7), ALDH activity (-49%), and telomerase activity (-47%). Therefore, age is associated with declining proliferative potential and regenerative functions of LMSCs in the lung.

Age-dependent decline in mouse lung regeneration with loss of lung fibroblast clonogenicity and increased myofibroblastic differentiation.

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.

Lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of elastase-injured lung.

While multipotent mesenchymal stromal cells have been recently isolated from adult lung (L-MSCs), there is very limited data on their biological properties and therapeutic potential in vivo. How L-MSCs compare with bone marrow-derived MSCs (BM-MSCs) is also unclear. In this study, we characterized L-MSC phenotype, clonogenicity, and differentiation potential, and compared L-MSCs to BM-MSCs in vivo survival, retention, paracrine gene expression, and repair or elastase injury after transplantation. L-MSCs were highly clonogenic, frequently expressed aldehyde dehydrogenase activity, and differentiated into osteocytes, chondrocytes, adipocytes, myofibroblasts, and smooth muscle cells. After intravenous injection (2 h), L-MSCs showed greater survival than BM-MSCs; similarly, L-MSCs were significantly more resistant than BM-MSCs to anchorage independent culture (4 h) in vitro. Long after transplantation (4 or 32 days), a significantly higher number of CD45(neg) L-MSCs were retained than BM-MSCs. By flow cytometry, L-MSCs expressed more intercellular adhesion molecule-1 (ICAM-1), platelet derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs; these proteins were found to modulate endothelial adherence, directional migration, and migration across Matrigel in L-MSCs. Further, L-MSCs with low ICAM-1 showed poorer lung retention and higher phagocytosis in vivo. Compared with BM-MSCs, L-MSCs expressed higher levels of several transcripts (e.g., Ccl2, Cxcl2, Cxcl10, IL-6, IL-11, Hgf, and Igf2) in vitro, although gene expression in vivo was increased by L-MSCs and BM-MSCs equivalently. Accordingly, both L-MSCs and BM-MSCs reduced elastase injury to the same extent. This study demonstrates that tissue-specific L-MSCs possess mechanisms that enhance their lung retention after intravenous transplantation, and produce substantial healing of elastase injury comparable to BM-MSCs.