GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Mesenchymal stem cell-derived extracellular vesicles in skin wound healing: roles, opportunities and challenges.

Skin wounds are characterized by injury to the skin due to trauma, tearing, cuts, or contusions. As such injuries are common to all human groups, they may at times represent a serious socioeconomic burden. Currently, increasing numbers of studies have focused on the role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in skin wound repair. As a cell-free therapy, MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy. Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures, including the regeneration of vessels, nerves, and hair follicles. In addition, MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization, wound angiogenesis, cell proliferation, and cell migration, and by inhibiting excessive extracellular matrix production. Additionally, these structures can serve as a scaffold for components used in wound repair, and they can be developed into bioengineered EVs to support trauma repair. Through the formulation of standardized culture, isolation, purification, and drug delivery strategies, exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair. In conclusion, MSC-derived EVs-based therapies have important application prospects in wound repair. Here we provide a comprehensive overview of their current status, application potential, and associated drawbacks.

GLI1+ cells are a source of repair-supportive mesenchymal cells (RSMCs) during airway epithelial regeneration.

Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.

Evidence for Multiple Origins of De Novo Formed Vascular Smooth Muscle Cells in Pulmonary Hypertension: Challenging the Dominant Model of Pre-Existing Smooth Muscle Expansion.

Vascular remodeling is a prominent feature of pulmonary hypertension. This process involves increased muscularization of already muscularized vessels as well as neo-muscularization of non-muscularized vessels. The cell-of-origin of the newly formed vascular smooth muscle cells has been a subject of intense debate in recent years. Identifying these cells may have important clinical implications since it opens the door for attempts to therapeutically target the progenitor cells and/or reverse the differentiation of their progeny. In this context, the dominant model is that these cells derive from pre-existing smooth muscle cells that are activated in response to injury. In this mini review, we present the evidence that is in favor of this model and, at the same time, highlight other studies indicating that there are alternative cellular sources of vascular smooth muscle cells in pulmonary vascular remodeling.

Validation of a Novel Fgf10 Cre-ERT2 Knock-in Mouse Line Targeting FGF10Pos Cells Postnatally.

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10 Cre-ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10 Ki-v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10 Cre-ERT2 line (called thereafter Fgf10 Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10 Ki-v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10 Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10 Ki-v2 mice. Bioinformatic analysis shows that the 3'UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10 Ki-v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10 Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.

Identification of a Repair-Supportive Mesenchymal Cell Population during Airway Epithelial Regeneration.

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα+ cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.

Targeting Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension (BPD-PH): Potential Role of the FGF Signaling Pathway in the Development of the Pulmonary Vascular System.

More than 50 years after the first description of Bronchopulmonary dysplasia (BPD) by Northway, this chronic lung disease affecting many preterm infants is still poorly understood. Additonally, approximately 40% of preterm infants suffering from severe BPD also suffer from Bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH), leading to a significant increase in total morbidity and mortality. Until today, there is no curative therapy for both BPD and BPD-PH available. It has become increasingly evident that growth factors are playing a central role in normal and pathologic development of the pulmonary vasculature. Thus, this review aims to summarize the recent evidence in our understanding of BPD-PH from a basic scientific point of view, focusing on the potential role of Fibroblast Growth Factor (FGF)/FGF10 signaling pathway contributing to disease development, progression and resolution.

Evidence for lung repair and regeneration in humans: key stem cells and therapeutic functions of fibroblast growth factors.

Regeneration carries the idea of regrowing partially or completely a missing organ. Repair, on the other hand, allows restoring the function of an existing but failing organ. The recognition that human lungs can both repair and regenerate is quite novel, the concept has not been widely used to treat patients. We present evidence that the human adult lung does repair and regenerate and introduce different ways to harness this power. Various types of lung stem cells are capable of proliferating and differentiating upon injury driving the repair/regeneration process. Injury models, primarily in mice, combined with lineage tracing studies, have allowed the identification of these important cells. Some of these cells, such as basal cells, broncho-alveolar stem cells, and alveolar type 2 cells, rely on fibroblast growth factor (FGF) signaling for their survival, proliferation and/or differentiation. While preclinical studies have shown the therapeutic benefits of FGFs, a recent clinical trial for acute respiratory distress syndrome (ARDS) using intravenous injection of FGF7 did not report the expected beneficial effects. We discuss the potential reasons for these negative results and propose the rationale for new approaches for future clinical trials, such as delivery of FGFs to the damaged lungs through efficient inhalation systems, which may be more promising than systemic exposure to FGFs. While this change in the administration route presents a challenge, the therapeutic promises displayed by FGFs are worth the effort.

Metformin induces lipogenic differentiation in myofibroblasts to reverse lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFß1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.

Role of Fibroblast Growth Factor 10 in Mesenchymal Cell Differentiation During Lung Development and Disease.

During organogenesis and pathogenesis, fibroblast growth factor 10 (Fgf10) regulates mesenchymal cell differentiation in the lung. Different cell types reside in the developing lung mesenchyme. Lineage tracing in vivo was used to characterize these cells during development and disease. Fgf10-positive cells in the early lung mesenchyme differentiate into multiple lineages including smooth muscle cells (SMCs), lipofibroblasts (LIFs) as well as other cells, which still remain to be characterized. Fgf10 signaling has been reported to act both in an autocrine and paracrine fashion. Autocrine Fgf10 signaling is important for the differentiation of LIF progenitors. Interestingly, autocrine Fgf10 signaling also controls the differentiation of pre-adipocytes into mature adipocytes. As the mechanism of action of Fgf10 on adipocyte differentiation via the activation of peroxisome proliferator-activated receptor gamma (Pparγ) signaling is quite well established, this knowledge could be instrumental for identifying drugs capable of sustaining LIF differentiation in the context of lung injury. We propose that enhanced LIF differentiation could be associated with improved repair. On the other hand, paracrine signaling is considered to be critical for the differentiation of alveolar epithelial progenitors during development as well as for the maintenance of the alveolar type 2 (AT2) stem cells during homeostasis. Alveolar myofibroblasts (MYFs), which are another type of mesenchymal cells critical for the process of alveologenesis (the last phase of lung development) express high levels of Fgf10 and are also dependent for their formation on Fgf signaling. The characterization of the progenitors of alveolar MYFs as well the mechanisms involved in their differentiation is paramount as these cells are considered to be critical for lung regeneration. Finally, lineage tracing in the context of lung fibrosis demonstrated a reversible differentiation from LIF to "activated" MYF during fibrosis formation and resolution. FGF10 expression in the lungs of idiopathic pulmonary fibrosis (IPF) vs. donors as well as progressive vs. stable IPF patients supports our conclusion that FGF10 deficiency could be causative for IPF progression. The therapeutic application of recombinant human FGF10 is therefore very promising.

miR-200b Mediates Endothelial-to-Mesenchymal Transition in Diabetic Cardiomyopathy.

Hyperglycemia-induced endothelial injury is a key pathogenetic factor in diabetic cardiomyopathy. Endothelial injury may lead to a phenotypic change (i.e., endothelial-to-mesenchymal transition [EndMT]), causing cardiac fibrosis. Epigenetic mechanisms, through specific microRNA, may regulate such a process. We investigated the mechanisms for such changes in cardiac microvascular endothelial cells and in the heart of genetically engineered mice with chemically induced diabetes. Cardiac tissues and isolated mouse heart endothelial cells (MHECs) from animals with or without endothelial-specific overexpression of miR-200b, with or without streptozotocin-induced diabetes, were examined at the mRNA and protein levels for endothelial and mesenchymal markers. Expression of miR-200b and its targets was quantified. Cardiac functions and structures were analyzed. In the hearts of wild-type diabetic mice, EndMT was observed, which was prevented in the miR-200b transgenic diabetic mice. Expression of specific markers such as vascular endothelial growth factor, zinc finger E-box-binding homeobox, transforming growth factor-ß1, and p300 were increased in the hearts of diabetic mice and were prevented following miR-200b overexpression. MHECs showed similar changes. miR-200b overexpression also prevented diabetes-induced cardiac functional and structural changes. These data indicate that glucose-induced EndMT in vivo and in vitro in the hearts of diabetic mice is possibly mediated by miR-200b and p300.