Fibroblast and myofibroblast activation in normal tissue repair and fibrosis.

The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Elevated numbers of infiltrating eosinophils accelerate the progression of Duchenne muscular dystrophy pathology in mdx mice.

Eosinophils, best known for their role in anti-parasitic responses, have recently been shown to actively participate in tissue homeostasis and repair. Their regulation must be tightly controlled, as their absence or hyperplasia is associated with chronic disease (e.g. asthma or inflammatory bowel disease). In the context of skeletal muscle, eosinophils play a supportive role after acute damage. Indeed, their depletion leads to strong defects in skeletal muscle regeneration and, in the absence of eosinophil-secreted interleukin (IL) 4 and IL13, fibro-adipogenic progenitors fail to support muscle stem cell proliferation. However, the role of eosinophils in muscular dystrophy remains elusive. Although it has been shown that eosinophils are present in higher numbers in muscles from mdx mice (a mouse model for Duchenne muscular dystrophy), their depletion does not affect muscle histopathology at an early age. Here, we evaluated the impact of hyper-eosinophilia on the development of fibrofatty infiltration in aged mdx mice and found that muscle eosinophilia leads to defects in muscle homeostasis, regeneration and repair, and eventually hastens death.

TNFα and IFNγ cooperate for efficient pro- to anti-inflammatory transition of macrophages during muscle regeneration.

IFNγ is traditionally known as a proinflammatory cytokine with diverse roles in antimicrobial and antitumor immunity. Yet, findings regarding its sources and functions during the regeneration process following a sterile injury are conflicting. Here, we show that natural killer (NK) cells are the main source of IFNγ in regenerating muscle. Beyond this cell population, IFNγ production is limited to a small population of T cells. We further show that NK cells do not play a major role in muscle regeneration following an acute injury or in dystrophic mice. Surprisingly, the absence of IFNγ per se also has no effect on muscle regeneration following an acute injury. However, the role of IFNγ is partially unmasked when TNFα is also neutralized, suggesting a compensatory mechanism. Using transgenic mice, we showed that conditional inhibition of IFNGR1 signaling in muscle stem cells or fibro-adipogenic progenitors does not play a major role in muscle regeneration. In contrast to common belief, we found that IFNγ is not present in the early inflammatory phase of the regeneration process but rather peaks when macrophages are acquiring an anti-inflammatory phenotype. Further transcriptomic analysis suggests that IFNγ cooperates with TNFα to regulate the transition of macrophages from pro- to anti-inflammatory states. The absence of the cooperative effect of these cytokines on macrophages, however, does not result in significant regeneration impairment likely due to the presence of other compensatory mechanisms. Our findings support the arising view of IFNγ as a pleiotropic inflammatory regulator rather than an inducer of the inflammatory response.

The paradigm shift in treatment of severe venous thromboembolism.

Pulmonary embolism (PE) is the third leading cause of cardiovascular death and the main cause of preventable in-hospital death in the world. The PERT® (Pulmonary Embolism Response Team) concept involves multidisciplinary diagnosis and immediate treatment. Deep venous thrombosis (DVT) is the initial cause of most cases of PE and is responsible for complications such as chronic thromboembolic recurrence, postthrombotic syndrome, and chronic thromboembolic pulmonary hypertension. An aggressive approach to severe cases of iliofemoral DVT similar to the PERT® system can not only reduce the immediate risk of PE and death but can also reduce later sequelae. New percutaneous techniques and mechanical thrombectomy devices for venous thromboembolism (VTE) have shown encouraging clinical results. We propose the development of an expanded concept of rapid response to VTE, which involves not only PE (PERT®) but also severe cases of DVT: the Venous Thromboembolism Response Team (VTERT®).

Efficacy and safety of iliofemoral bypass using arm veins as an alternative conduit for chronic limb-threatening ischemia.

OBJECTIVE: The use of basilic vein in iliofemoral revascularizations was previously described in the literature as an autologous option for the treatment of vascular prosthesis infection and as a primary conduit in patients at high risk of infectious surgical complications. However, the publications available include several different indications and are limited to case reports. Therefore, the aim of this study was to evaluate the outcomes of the use of arm veins as a safe and effective autologous alternative for iliofemoral reconstruction in patients with chronic limb-threatening ischemia (CLTI) and at high risk of prosthesis infection. METHODS: We performed a multicenter, retrospective cohort study with 53 consecutive iliofemoral bypasses using arm veins as an alternative conduit. The procedures were performed between November 2013 and November 2021, exclusively for patients with CLTI classified as TASC aortoiliac C or D with increased risk of postoperative surgical infection. Demographic, clinical variables, and outcomes were collected from a prospective database. Main endpoints were amputation-free survival (AFS) and major adverse cardiovascular events. Secondary endpoints included primary and secondary patencies and overall survival. Cox regression analysis was used to identify the predictors of AFS. Postoperative surgical complications and 30-day mortality were also assessed. RESULTS: The mean age was 64.2 ± 8.4 years, with a predominance of male gender. The median follow-up period was 615 days. All patients had CLTI, with a predominance of tissue loss (n = 51; 96.2%) and a median ankle-brachial index of 0.28. The basilic vein was utilized in most procedures (69.8%). Thirty-day major adverse cardiovascular events occurred in five cases (9.4%), and the 30-day mortality rate was 3.8%. The AFS, primary patency, secondary patency, and overall survival in 720 days were 71%, 72%, 89%, and 75%, respectively. Cox regression analysis revealed no association between the variables analyzed for AFS. There was no graft late infection nor pseudoaneurysmal degeneration. CONCLUSIONS: Iliofemoral bypass using arm veins as an autologous conduit proved to be an effective and safe procedure with low incidence of postoperative cardiovascular complications and high rates of AFS in patients with CLTI. Also, this suggests that arm veins can be an interesting and suitable autologous alternative conduit for iliofemoral reconstructions, especially in cases in which a prosthesis should be avoided or when it is not available.

High anti-tumor activity of a novel alpha-fetoprotein-maytansinoid conjugate targeting alpha-fetoprotein receptors in colorectal cancer xenograft model.

The alpha-fetoprotein receptor (AFPR) is a novel target for cancer therapeutics. It is expressed on most cancers and myeloid derived suppressor cells (MDSCs) but generally absent on normal tissues. Studies were performed to investigate the use of recombinant human AFP (ACT-101) conjugated with maytansinoid toxins for targeted toxin delivery to cancer. Four structurally different ACT-101-maytansinoid conjugates containing cleavable glutathione sensitive linkers were initially investigated in a mouse xenograft model of colorectal cancer. Reduction in tumor volume was seen for all four conjugates compared to control (p < 0.05). The anti-tumor effects of the conjugate selected for further development (ACT-903) persisted after treatment discontinuation, with tumors becoming undetectable in 9 of 10 mice, and all 10 mice surviving through Day 60 with no obvious signs of toxicity. A follow-up study performed in the same model compared the effects of single intravenous doses of ACT-903 (10-50 mg/kg) to that of control groups receiving vehicle or ACT-101. A significant reduction of tumor burden compared to control was achieved in the 40 and 50 mg/kg dose groups. Survival was significantly prolonged in these 2 groups (40 mg/kg (p < 0.0001); 50 mg/kg (p = 0.0037). Free maytansine blood levels at 4 h were 0.008% of the dose, indicating stability of the conjugate in circulation as was expected based on in vitro plasma stability studies. No obvious signs of toxicity were seen in any of the treated groups. Observed efficacy and excellent tolerability of ACT-903 in these xenograft models support advancing the development of ACT-903 toward clinical use.

Emerging skeletal muscle stromal cell diversity: Functional divergence in fibro/adipogenic progenitor and mural cell populations.

While the majority of healthy skeletal muscle consists of multinucleated syncytial repetitive contractile myofibers, repaired by skeletal muscle stem cells when damaged, the maintenance of muscle function also requires a range of tissue-resident stromal populations. In fact, the careful orchestration of damage response processes upon muscle injury relies heavily on stromal cell contribution for effective repair. The two main types of muscle-resident stromal cells are fibro/adipogenic progenitors and mural cells. The latter is comprised of pericytes and vascular smooth muscle cells. Recent publications identifying common markers for stromal cell populations have allowed investigating population dynamics throughout the regenerative process at a higher resolution. Mounting evidence now suggests that subpopulations with distinct roles may exist among stromal cells. In various degenerative muscle wasting conditions, critical cross-talk and spatial signalling amongst various cell populations become dysregulated. This can result in the failure to curb pathological fibro/adipogenic progenitor proliferation and propensity for laying down excessive extracellular matrix, which in turn leads to fibrotic infiltration, reduced contractile units and gradual decline in muscle function. Restoration of physiologically appropriate stromal cell function is therefore just as crucial for therapeutic targeting as the homeostatic maintenance of muscle function.

Lipid nanoparticle-mediated silencing of osteogenic suppressor GNAS leads to osteogenic differentiation of mesenchymal stem cells in vivo.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest. Here we examine the ability of small interfering RNA (siRNA) to enhance osteoblast differentiation and bone formation by silencing the negative suppressor gene GNAS in bone MSCs. Using clinically validated lipid nanoparticle (LNP) siRNA delivery systems, we show that silencing the suppressor gene GNAS in vitro in MSCs leads to molecular and phenotypic changes similar to those seen in osteoblasts. Further, we demonstrate that these LNP-siRNAs can transfect a large proportion of mice MSCs in the compact bone following intravenous injection. Transfection of MSCs in various animal models led to silencing of GNAS and enhanced differentiation of MSCs into osteoblasts. These data demonstrate the potential for LNP delivery of siRNA to enhance the differentiation of MSCs into osteoblasts, and suggests that they are a promising approach for the treatment of osteoporosis and other bone diseases.

The Gamma and Neutron Sensor System for Rapid Dose Rate Mapping in the CLEANDEM Project.

The decommissioning of nuclear installations, as well as the possible necessary accident remediations, requires the physical presence of human operators in potentially radiologically hostile environments. The number of active nuclear reactors worldwide is greater than 400, and most of them are 40 to 50 years old, thus implying that soon they will have to be dismantled. In the framework of the H2020 CLEANDEM project, a small robotic vehicle is being developed that is equipped with a series of different sensors for areas that are significantly contaminated by radiation. In this work, we describe the MiniRadMeter system, a compact low-cost sensor capable of being used to perform quick gamma and neutron radiation field mapping of environments prior to the possible start of human operations. The miniature gamma sensor is a 1 cm3 scintillator counter with moderate spectroscopic features read out by means of a 6 × 6 mm2 SiPM, whereas neutrons are detected by means of a silicon diode coupled to a layer of 6LiF and placed inside a 6 × 6 × 6 cm3 polyethylene box. The front-end and data acquisition electronics were developed based on a Raspberry Pi4 microcomputer. In this paper, the system performance and the preliminary test results are described.

Management of traumatic superficial palmar arch pseudoaneurysm: a therapeutic challenge.

Pseudoaneurysm of the palmar arch is a rare entity. Diagnosis is dependent on high clinical suspicion. We present a case referred to the emergency department, with a history of glass penetrating trauma to the palmar surface with a pulsatile mass and jet bleeding. Doppler ultrasound evidenced a partially thrombosed pseudoaneurysm. A CT angiography examination showed a saccular formation arising from the superficial palmar arch. A conventional surgical approach was indicated. A clinical suspicion must be ventured to arrive at the correct diagnosis. Imaging modalities are needed to identify the pseudoaneurysm and plan the treatment course. Nonetheless, the sequence of diagnosis is individual, because further evaluation with different imaging methods may not change the rationale for the intervention. In our experience, conventional surgical removal is preferable, due to its safety and well-established outcomes.

O pseudoaneurisma do arco palmar é uma entidade rara, cujo diagnóstico depende de alta suspeição clínica. Apresentamos o caso de um paciente encaminhado ao pronto-socorro com história de traumatismo penetrante por vidro na face palmar, com massa pulsátil e sangramento em jato. A ultrassonografia com Doppler evidenciou pseudoaneurisma parcialmente trombosado, e a angiotomografia demonstrou formação sacular originada do arco palmar superficial. Uma abordagem cirúrgica convencional foi indicada. Para prosseguir com o diagnóstico correto, essa suspeita clínica deve ser aventada. Modalidades de imagem são necessárias para identificar o pseudoaneurisma e planejar o curso do tratamento. No entanto, a sequência diagnóstica é individual, pois uma avaliação mais aprofundada, com diferentes métodos de imagem, pode não alterar o racional da intervenção. Em nossa experiência, a remoção cirúrgica convencional é preferível, visto sua segurança e seus resultados bem estabelecidos.

TGF-ß-driven downregulation of the transcription factor TCF7L2 affects Wnt/ß-catenin signaling in PDGFRα+ fibroblasts.

Mesenchymal stromal cells (MSCs) are multipotent progenitors essential for organogenesis, tissue homeostasis, regeneration and scar formation. Tissue injury upregulates transforming growth factor ß (TGF-ß) signaling, which modulates myofibroblast fate, extracellular matrix remodeling and fibrosis. However, the molecular determinants of MSC differentiation and survival remain poorly understood. During canonical Wnt signaling, T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors regulate development and stemness, but the mechanisms by which injury-induced cues modulate their expression remain underexplored. Here, we studied the cell type-specific gene expression of TCF/LEF transcription factors and, more specifically, we investigated whether damage-induced TGF-ß signaling impairs the expression and function of TCF7L2 (also known as TCF4), using several models of MSCs, including skeletal muscle fibro-adipogenic progenitors. We show that TCF/LEFs are differentially expressed and that TGF-ß reduces the expression of TCF7L2 in MSCs but not in myoblasts. We also found that the ubiquitin-proteasome system regulates TCF7L2 proteostasis and participates in TGF-ß-mediated TCF7L2 protein downregulation. Finally, we show that TGF-ß requires histone deacetylase activity to repress the expression of TCF7L2. Thus, our work reports a novel interplay between TGF-ß and canonical Wnt signaling cascades in PDGFRα+ fibroblasts and suggests that this mechanism could be targeted in tissue repair and regeneration.

The origins and non-canonical functions of macrophages in development and regeneration.

The discovery of new non-canonical (i.e. non-innate immune) functions of macrophages has been a recurring theme over the past 20 years. Indeed, it has emerged that macrophages can influence the development, homeostasis, maintenance and regeneration of many tissues and organs, including skeletal muscle, cardiac muscle, the brain and the liver, in part by acting directly on tissue-resident stem cells. In addition, macrophages play crucial roles in diseases such as obesity-associated diabetes or cancers. Increased knowledge of their regulatory roles within each tissue will therefore help us to better understand the full extent of their functions and could highlight new mechanisms modulating disease pathogenesis. In this Review, we discuss recent studies that have elucidated the developmental origins of various macrophage populations and summarize our knowledge of the non-canonical functions of macrophages in development, regeneration and tissue repair.

An original amino acid formula favours in vitro corneal epithelial wound healing by promoting Fn1, ITGB1, and PGC-1α expression.

Corneal disorders are frequent, involving most diabetic patients; among its manifestations, they include delayed wound healing. Since maintenance of mitochondrial homeostasis is fundamental for the cell, stimulation of mitochondrial biogenesis represents a unique therapeutic tool for preventing and treating disorders with a deficit in energy metabolism. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem) supported mitochondrial biogenesis in cardiac and skeletal muscle, reduced liver damage caused by alcohol, and prevented the doxorubicin-dependent mitochondrial damage in cardiomyocytes. The present study aimed to investigate a new amino acid mixture, named six amino acids (6AA), to promote corneal epithelial wound healing by regulating mitochondrial biogenesis. A murine epithelium cell line (TKE2) exposed to this mixture showed increased mitochondrial biogenesis markers, fibronectin 1 (Fn1) and integrin beta 1 (ITGB1) involved in extracellular matrix synthesis and cell migration. Most importantly, the 6AA mixture completely restored the wound in scratch assays, confirming the potential of this new formula in eye disorders like keratopathy. Moreover, our results demonstrate for the first time that peroxisome proliferator-receptor γ coactivator 1 α (PGC-1α) is expressed in TKE2 cells, which controls mitochondrial function and corneal repair process. These results could be relevant for the treatment mainly focused on corneal re-epithelialisation.

Thermoneutral Housing and a Western Diet Combination Exacerbates Dysferlin-Deficient Muscular Dystrophy.

INTRODUCTION/AIMS: Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). METHODS: The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. RESULTS: BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. DISCUSSION: TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.

Real Life Outcomes of Infrapopliteal Endovascular and Surgical Bypass Intervention for Chronic Limb Threatening Ischaemia in GLASS Stages II and III.

OBJECTIVE: Great efforts have been made to choose between bypass surgery and angioplasty as the first choice for revascularisation in chronic limb threatening ischaemia (CLTI). Endovascular therapy predominates despite limited evidence for its advantages. The purpose of this observational cohort study was to investigate outcomes after open and endovascular infrapopliteal revascularisation in extensive infrainguinal arterial disease. METHODS: The medical records of 1 427 patients who underwent infrainguinal revascularisation exclusively for CLTI in the period January 2014 to February 2019 were reviewed. After detailed analysis, only infrapopliteal revascularisations classified as GLASS stage II or III were considered, resulting in 326 procedures. In total, 127 patients underwent endovascular therapy and 199 patients underwent bypass graft surgery (BGS). The primary endpoints included amputation free survival (AFS) and overall survival (OS). Secondary endpoints included the analyses of multiple factors related to long term AFS. RESULTS: Regarding the primary endpoint, AFS was 75.2% and 65.2% at one and three years, respectively. OS at one and three years was 91.2% and 83.1%, respectively. In the univariable analysis, the hazard of the combined endpoint of major amputation or death was higher after bypass surgery than after endovascular therapy (hazard ratio [HR] 1.80, 95% confidence interval [CI] 1.13 - 2.89; p = .013). After either revascularisation method, TASC II femoropopliteal D was associated with a higher risk of amputation or death (HR 1.69, 95% CI 1.10 - 2.58; p = .015). Multivariable Cox regression analysis revealed no association between the variables analysed for AFS. CONCLUSION: Patients with CLTI submitted to infrapopliteal revascularisation and classified as GLASS II and III had satisfactory AFS and OS rates after an individualised team conference decision. Furthermore, the revascularisation modality (endovascular or open) did not influence the AFS results.

A designer mixture of six amino acids promotes the extracellular matrix gene expression in cultured human fibroblasts.

The deterioration of the skin is caused by dermatological disorders, environmental conditions, and aging processes. One incisive strategy for supervising the skin aging process is implementing healthy nutrition, preserving a balanced diet, and a good supply of food supplements. Here, we compared H-Pro-Hyp-OH peptide, hydrolyzed collagen, and an original mixture of six amino acids (we named 6aa)-including glycine, l-alanine, l-proline, l-valine, l-leucine, and l-lysine-effects on the production of extracellular matrix (ECM) components, particularly the elastin, fibronectin, collagen 1, and collagen 4. Treatment of BJ human skin fibroblasts with the 6aa mixture upregulated elastin, fibronectin, and collagen 1 gene expression, without affecting the expression of anti-reactive oxygen species enzymes. Moreover, the mammalian target of rapamycin (mTOR) signaling pathway seems to be involved, at least in part. Collectively, these results suggest that the six amino acid mixture exerts beneficial effects in human skin fibroblasts.

The methyltransferase G9a regulates HoxA9-dependent transcription in AML.

Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.