MSC-NTF (NurOwn®) exosomes: a novel therapeutic modality in the mouse LPS-induced ARDS model.

BACKGROUND: One of the most severe complications of the current COVID-19 pandemic is acute respiratory distress syndrome (ARDS). ARDS is caused by increased amounts of pro-inflammatory cytokines, leading to lung damage and loss of lung function. There are currently no effective therapies for combatting ARDS. Mesenchymal stem cells (MSCs) have been suggested as a potential treatment for ARDS due to their significant immunomodulatory properties. MSC small extracellular vesicles (sEVs), including exosomes, modulate the immune response as effectively as MSCs themselves, with the added advantages of increased safety and tissue penetration. METHODS: We isolated sEVs from MSCs induced to secrete increased levels of neurotrophic and immunomodulatory factors, termed Exo MSC-NTF, and compared their ability to treat ARDS, in a lung injury LPS mouse model, to sEVs isolated from naïve MSCs (Exo MSC). Measurments of lung histopathological changes and neutrophil infiltration, blood oxygen saturation, and bronchoalveolar lavge fluid (BALF) proinflammatory cytokines and coagulation related factors were performed. RESULTS: We found that Exo MSC-NTF was superior to Exo MSC in reducing LPS-induced ARDS markers, including physiological lung damage such as alveolar wall thickness, fibrin presence, and neutrophil accumulation, as well as increasing oxygenation levels. Furthermore, Exo MSC-NTF reversed the imbalance in the host immune response, seen as decreased IFN-γ, IL-6, TNF-α, and RANTES levels in the bronchoalveolar lavage fluid. CONCLUSIONS: These positive preclinical results suggest that Exo MSC-NTF may be suitable as a therapy for COVID-19-induced ARDS and are more effective at combatting ARDS physiological, pathological, and biochemical symptoms than sEVs isolated from non-induced MSCs.

Deletion of the Mir-106b~ 25 MicroRNA cluster attenuates atherosclerosis in Apolipoprotein E knockout mice.

BACKGROUND: MicroRNAs are short non-coding RNAs that regulate gene expression. The aim of this study was to gain an understanding of the possible role of the miR-106b~ 25 microRNA cluster in regulating atherosclerosis in mice. METHODS: MiR-106b~ 25 knockout mice were outcrossed into Apolipoprotein E (ApoE) knockout background to generate double knockout mice. At 36 weeks of age, lesion size was evaluated in the aortic sinus by oil-red-O staining. RESULTS: Lesion size was 2-fold smaller in double KO mice in comparison to ApoE KO mice. In addition, collagen staining showed a trend towards a stable plaque phenotype in the double KO mice. Lipid profiling of plasma samples of double KO and ApoE KO mice using FPLC revealed over 2-fold decrease in Very low density lipoprotein (VLDL) cholesterol content and a 50% decrease in low density lipoprotein (LDL) cholesterol content in double KO mice. By using target prediction software, we have identified several possible targets for the miR-106b~ 25 cluster including the VLDL and LDL receptors. We found that upon feeding miR-106b~ 25 KO mice with high fat diet, the expression of LDL and VLDL receptors was higher than in the wild-type mice, suggesting the miR-106b~ 25 cluster regulates atherosclerosis by influencing clearance of VLDL and LDL from the plasma. CONCLUSIONS: We identified the miR-106b~ 25 cluster as a novel regulator of atherosclerosis in ApoE KO mice, presumably by regulating plasma cholesterol levels.

Distinct origins and molecular mechanisms contribute to lymphatic formation during cardiac growth and regeneration.

In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.

Development of the lymphatic system: new questions and paradigms.

The lymphatic system is a blind-ended network of vessels that plays important roles in mediating tissue fluid homeostasis, intestinal lipid absorption and the immune response. A profound understanding of the development of lymphatic vessels, as well as of the molecular cues governing their formation and morphogenesis, might prove essential for our ability to treat lymphatic-related diseases. The embryonic origins of lymphatic vessels have been debated for over a century, with a model claiming a venous origin for the lymphatic endothelium being predominant. However, recent studies have provided new insights into the origins of lymphatic vessels. Here, we review the molecular mechanisms controlling lymphatic specification and sprouting, and we discuss exciting findings that shed new light on previously uncharacterized sources of lymphatic endothelial cells.

Experimental myocardial infarction induces altered regulatory T cell hemostasis, and adoptive transfer attenuates subsequent remodeling.

BACKGROUND: Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling. METHODS AND RESULTS: The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling. CONCLUSION: Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.

The transient receptor potential vanilloid 2 cation channel is abundant in macrophages accumulating at the peri-infarct zone and may enhance their migration capacity towards injured cardiomyocytes following myocardial infarction.

PURPOSE: A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3-5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI. METHODS: Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofluorescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted. RESULTS: The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3-5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced. CONCLUSIONS: TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions.

Regulatory T cells influence blood flow recovery in experimental hindlimb ischaemia in an IL-10-dependent manner.

AIMS: Ischaemic damage is associated with up-regulation of pro-inflammatory cytokines, as well as invasion of leucocytes and lymphocytes to the injured muscle. Regulatory T cells (Tregs) exert suppressive effects on several immune and non-immune cellular elements. We hypothesized that adoptive Treg cell transfer and depletion will influence re-establishment of flow in the hindlimb ischaemia model, and that this effect would be mediated by the cytokine interleukin (IL)-10. METHODS AND RESULTS: To study the functional role of Tregs in hindlimb ischaemia, we either adoptively transferred Tregs or functionally blocked Tregs by antibodies to CD25. Initially, we showed that the number and function of Tregs is altered after the induction of ischaemia. Treg ablation resulted in reduced blood flow by laser Doppler at Day 7 that became more robust at Day 14. Adoptive Treg transfer led to a significant improvement of flow in the ligated limb. Treg-mediated improvement in flow was abolished by employing blocking anti-IL-10 antibodies. CONCLUSIONS: These results show that Tregs play an important role in processes that control flow re-establishment after inducible hindlimb ischaemia, and that IL-10 plays a requisite role mediating these effects.

The 106b∼25 microRNA cluster is essential for neovascularization after hindlimb ischaemia in mice.

AIMS: MicroRNAs (miRNAs, miR) are endogenous short RNA sequences that regulate a wide range of physiological and pathophysiological processes. Several miRNAs control the formation of new blood vessels either by increasing or by inhibiting angiogenesis. Here, we investigated the possible role of the miR-106b∼25 cluster in postnatal neovascularization and in regulation of the angiogenic properties of adult bone marrow-derived stromal cells. METHODS AND RESULTS: To study the effect of miR-106b∼25 deletion on neovascularization, we used a miR-106b∼25 knockout (KO) mouse model. After inducing hindlimb ischaemia, we showed that vascularization in ischaemic mice devoid of miR-106b∼25 is impaired, as evident from the reduced blood flow on laser Doppler perfusion imaging. The miR-106b∼25 cluster was also shown here to be an essential player in the proper functioning of bone marrow-derived stromal cells through its regulation of apoptosis, matrigel tube formation capacity, cytokine secretion, and expression of the stem-cell marker Sca-1. In addition, we showed that capillary sprouting from miR-106b∼25 KO aortic rings is diminished. CONCLUSION: These results show that the miR-106b∼25 cluster regulates post-ischaemic neovascularization in mice, and that it does so in part by regulating the function of angiogenic bone marrow-derived stromal cells and of endothelial cells.