Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse under various circumstances, but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant-cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas.

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies.

Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1iΔEC ) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/αvβ3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the αvβ3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease.

Senescent cells accumulate in multiple aging-associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal β-galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto-oligosaccharides. We show that gal-encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy-induced senescence, gal-encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal-encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal-encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders.

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.

Nanotechnology changed the concept of treatment for a variety of diseases, producing a huge impact regarding drug and gene delivery. Among the different targeted diseases, osteoporosis has devastating clinical and economic consequences. Since current osteoporosis treatments present several side effects, new treatment approaches are needed. Recently, the application of small interfering RNA (siRNA) has become a promising alternative. Wnt/β-catenin signaling pathway controls bone development and formation. This pathway is negatively regulated by sclerostin, which knock-down through siRNA application would potentially promote bone formation. However, the major bottleneck for siRNA-based treatments is the necessity of a delivery vector, bringing nanotechnology as a potential solution. Among the available nanocarriers, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of siRNAs. The mesoporous structure of MSNs permits the delivery of siRNAs together with another biomolecule, achieving a combination therapy. Here, the effectiveness of a new potential osteoporosis treatment based on MSNs is evaluated. The proposed system is effective in delivering SOST siRNA and osteostatin through systemic injection to bone tissue. The nanoparticle administration produced an increase expression of osteogenic related genes improving the bone microarchitecture. The treated osteoporotic mice recovered values of a healthy situation approaching to osteoporosis remission.

Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/β-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development.

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest cancers for which optimal diagnostic tools are still greatly needed. Identification of PDAC-specific molecular markers would be extremely useful to improve disease diagnosis and follow-up. MT1-MMP has long been involved in pancreatic cancer, especially in tumour invasion and metastasis. In this study, we aim to ascertain the suitability of MT1-MMP as a biomarker for positron emission tomography (PET) imaging. Two probes were assessed and compared for this purpose, an MT1-MMP-specific binding peptide (MT1-AF7p) and a specific antibody (LEM2/15), labelled, respectively, with 68Ga and with 89Zr. PET imaging with both probes was conducted in patient-derived xenograft (PDX), subcutaneous and orthotopic, PDAC mouse models, and in a cancer cell line (CAPAN-2)-derived xenograft (CDX) model. Both radiolabelled tracers were successful in identifying, by means of PET imaging techniques, tumour tissues expressing MT1-MMP although they did so at different uptake levels. The 89Zr-DFO-LEM2/15 probe showed greater specific activity compared to the 68Ga-labelled peptide. The mean value of tumour uptake for the 89Zr-DFO-LEM2/15 probe (5.67 ± 1.11%ID/g, n=28) was 25-30 times higher than that of the 68Ga-DOTA-AF7p ones. Tumour/blood ratios (1.13 ± 0.51 and 1.44 ± 0.43 at 5 and 7 days of 89Zr-DFO-LEM2/15 after injection) were higher than those estimated for 68Ga-DOTA-AF7p probes (of approximately tumour/blood ratio = 0.5 at 90 min after injection). Our findings strongly point out that (i) the in vivo detection of MT1-MMP by PET imaging is a promising strategy for PDAC diagnosis and (ii) labelled LEM2/15 antibody is a better candidate than MT1-AF7p for PDAC detection.

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

MT1-matrix metalloproteinase (MT1-MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1-MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1-MMP occurs during MDA-MB-231 adenocarcinoma cell migration into collagen type I three-dimensional matrices. Polarized trafficking of MT1-MMP is triggered by beta1 integrin-mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1-MMP within VSV-G/Rab8-positive vesicles, but not in Rab11/Tf/TfRc-positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1-MMP exocytic traffic, collagen degradation and invasion, whereas Rab8- but not Rab11-knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1-MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1-MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis. Moreover, single-domain variable regions (VHHs or Nanobodies) derived from camelid heavy-chain-only antibodies have demonstrated improvements in tissue penetration and blood clearance, important characteristics for cancer imaging. Here, we have developed a nanobody-based PET imaging strategy for TNBC detection that targets MT1-MMP. A llama-derived library was screened against the catalytic domain of MT1-MMP and a panel of specific nanobodies were identified. After a deep characterization, two nanobodies were selected to be labeled with gallium-68 (68Ga). ImmunoPET imaging with both ([68Ga]Ga-NOTA-3TPA14 and [68Ga]Ga-NOTA-3CMP75) in a TNBC mouse model showed precise tumor-targeting capacity in vivo with high signal-to-background ratios. (68Ga)Ga-NOTA-3CMP75 exhibited higher tumor uptake compared to (68Ga)Ga-NOTA-3TPA14. Furthermore, imaging data correlated perfectly with the immunohistochemistry staining results. In conclusion, we found a promising candidate for nanobody-based PET imaging to be further investigated as a diagnostic tool in TNBC.

The vasculature ensures optimal delivery of nutrients and oxygen throughout the body, and to achieve this function it must continually adapt to varying tissue demands. Newly formed vascular plexuses during development are immature and require dynamic remodeling to generate well-patterned functional networks. This is achieved by remodeling of the capillaries preserving those which are functional and eliminating other ones. A balanced and dynamically regulated capillary remodeling will therefore ensure optimal distribution of blood and nutrients to the tissues. This is particularly important in pathological contexts in which deficient or excessive vascular remodeling may worsen tissue perfusion and hamper tissue repair. Blood flow is a major determinant of microvascular reshaping since capillaries are pruned when relatively less perfused and they split when exposed to high flow in order to shape the microvascular network for optimal tissue perfusion and oxygenation. The molecular machinery underlying blood flow sensing by endothelial cells is being deciphered, but much less is known about how this translates into endothelial cell responses as alignment, polarization and directed migration to drive capillary remodeling, particularly in vivo. Part of this knowledge is theoretical from computational models since blood flow hemodynamics are not easily recapitulated by in vitro or ex vivo approaches. Moreover, these events are difficult to visualize in vivo due to their infrequency and briefness. Studies had been limited to postnatal mouse retina and vascular beds in zebrafish but new tools as advanced microscopy and image analysis are strengthening our understanding of capillary remodeling. In this review we introduce the concept of remodeling of the microvasculature and its relevance in physiology and pathology. We summarize the current knowledge on the mechanisms contributing to capillary regression and to capillary splitting highlighting the key role of blood flow to orchestrate these processes. Finally, we comment the potential and possibilities that microfluidics offers to this field. Since capillary remodeling mechanisms are often reactivated in prevalent pathologies as cancer and cardiovascular disease, all this knowledge could be eventually used to improve the functionality of capillary networks in diseased tissues and promote their repair.

Introduction: Removal of poorly perfused capillaries by pruning contributes to remodeling the microvasculature to optimize oxygen and nutrient delivery. Blood flow drives this process by promoting the intravascular migration of endothelial cells in developing networks, such as in the yolk sac, zebrafish brain or postnatal mouse retina. Methods: In this study, we have implemented innovative tools to recognize capillary pruning in the complex 3D coronary microvasculature of the postnatal mouse heart. We have also experimentally tested the impact of decreasing pruning on the structure and function of this network by altering blood flow with two different vasodilators: losartan and prazosin. Results: Although both drugs reduced capillary pruning, a combination of experiments based on ex vivo imaging, proteomics, electron microscopy and in vivo functional approaches showed that losartan treatment resulted in an inefficient coronary network, reduced myocardial oxygenation and metabolic changes that delayed the arrest of cardiomyocyte proliferation, in contrast to the effects of prazosin, probably due to its concomitant promotion of capillary expansion. Discussion: Our work demonstrates that capillary pruning contributes to proper maturation and function of the heart and that manipulation of blood flow may be a novel strategy to refine the microvasculature and improve tissue perfusion after damage.

Heterogeneity and functional specialization among skin-resident macrophages are incompletely understood. In this study, we describe a novel subset of murine dermal perivascular macrophages that extend protrusions across the endothelial junctions in steady-state and capture blood-borne macromolecules. Unlike other skin-resident macrophages that are reconstituted by bone marrow-derived progenitors after a genotoxic insult, these cells are replenished by an extramedullary radio-resistant and UV-sensitive Bmi1(+) progenitor. Furthermore, they possess a distinctive anti-inflammatory transcriptional profile, which cannot be polarized under inflammatory conditions, and are involved in repair and remodeling functions for which other skin-resident macrophages appear dispensable. Based on all their properties, we define these macrophages as Skin Transendothelial Radio-resistant Anti-inflammatory Macrophages (STREAM) and postulate that their preservation is important for skin homeostasis.

Vascular smooth muscle cell (VSMC) proliferation is essential for arteriogenesis to restore blood flow after artery occlusion, but the mechanisms underlying this response remain unclear. Based on our previous findings showing increased VSMC proliferation in the neonatal aorta of mice lacking the protease MT4-MMP, we aimed at discovering new players in this process. We demonstrate that MT4-MMP absence boosted VSMC proliferation in vitro in response to PDGF-BB in a cell-autonomous manner through enhanced p38 MAPK activity. Increased phospho-p38 in basal MT4-MMP-null VSMCs augmented the rate of mitochondrial degradation by promoting mitochondrial morphological changes through the co-activator PGC1α as demonstrated in PGC1α-/- VSMCs. We tested the in vivo implications of this pathway in a novel conditional mouse line for selective MT4-MMP deletion in VSMCs and in mice pre-treated with the p38 MAPK activator anisomycin. Priming of p38 MAPK activity in vivo by the absence of the protease MT4-MMP or by anisomycin treatment led to enhanced arteriogenesis and improved flow recovery after femoral artery occlusion. These findings may open new therapeutic opportunities for peripheral vascular diseases.

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulatory properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Previous studies have demonstrated that MSCs, administered systemically, migrate to lymphoid tissues associated with the inflammatory site where functional MSC-induced immune cells with a regulatory phenotype were increased mediating the immunomodulatory effects of MSCs. These results suggest that homing of MSCs to the lymphatic system plays an important role in the mechanism of action of MSCs in vivo. Thus, we hypothesized that direct intralymphatic (IL) (also referred as intranodal) administration of MSCs could be an alternative and effective route of administration for MSC-based therapy. Here, we report the feasibility and efficacy of the IL administration of human expanded adipose mesenchymal stem cells (eASCs) in a mouse model of collagen-induced arthritis (CIA). IL administration of eASCs attenuated the severity and progression of arthritis, reduced bone destruction and increased the levels of regulatory T cells (CD25+Foxp3+CD4+ cells) and Tr1 cells (IL10+CD4+), in spleen and draining lymph nodes. Taken together, these results indicate that IL administration of eASCs is very effective in modulating established CIA and may represent an alternative treatment modality for cell therapy with eASCs.

Five-year survival for pancreatic ductal adenocarcinoma (PDAC) patients remains below 7% due to the lack of effective treatments. Here, we report that combined ablation of EGFR and c-RAF expression results in complete regression of a significant percentage of PDAC tumors driven by Kras/Trp53 mutations in genetically engineered mice. Moreover, systemic elimination of these targets induces toxicities that are well tolerated. Response to this targeted therapy correlates with transcriptional profiles that resemble those observed in human PDACs. Finally, inhibition of EGFR and c-RAF expression effectively blocked tumor progression in nine independent patient-derived xenografts carrying KRAS and TP53 mutations. These results open the door to the development of targeted therapies for PDAC patients.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.