Atherosclerosis remains the most important cause of death worldwide despite an extensive therapeutic arsenal. We previously showed that CD146, an adhesion molecule expressed by endothelial cells, is harbored by macrophages in atherosclerotic plaque and allows to reduce CCL5 and subsequent inflammation. As extracellular vesicles may serve as potential clinical delivery devices, we hypothesized that CD146 extracellular vesicles injection could be atheroprotective. We generated and purified extracellular vesicles from mouse endothelial cells deleted or not with CD146. In vitro stimulation of macrophages with CD146 extracellular vesicles induced macrophage polarization towards an anti-inflammatory phenotype through the STAT3/IL-10 axis, whereas CD146-negative extracellular vesicles did not have any effect. We next tracked the trafficking of labeled extracellular vesicles after intravenous injection in atherosclerotic ApoE-/- mice and visualized their incorporation into atheroma. After bi-weekly injections of extracellular vesicles for 6 weeks, only ApoE-/- mice treated with CD146 extracellular vesicles exhibited a significant reduction in atherosclerotic plaque, associated with an anti-inflammatory macrophage phenotype. We thus propose that treatment with CD146 extracellular vesicles could constitute a novel therapeutic option for reducing atherosclerosis.

RationaleCOVID-19-associated acute-respiratory distress syndrome (C-ARDS) results from a direct viral injury associated with host excessive innate immune response mainly affecting the lungs. However, cytokine profile in the lung compartment of C-ARDS patients has not been widely studied, nor compared to non-COVID related ARDS (NC-ARDS).ObjectivesTo evaluate caspase-1 activation, IL-1 signature, and other inflammatory cytokine pathways associated with tissue damage using post-mortem lung tissues, bronchoalveolar lavage fluids (BALF), and serum across the spectrum of COVID-19 severity.MethodsHistological features were described and activated-caspase-1 labeling was performed in 40 post-mortem biopsies. Inflammatory cytokines were quantified in BALF and serum from 19 steroid-treated-C-ARDSand compared to 19 NC-ARDS. Cytokine concentrations were also measured in serum from 128 COVID-19 patients at different severity stages.Measurements and main resultsTypical “diffuse alveolar damage” in lung biopsies were associated with activated caspase-1 expression and vascular lesions. Soluble Caspase-1p20, IL-1β, IL-1Ra, IL-6 and at lower level IFNγ and CXCL-10, were highly elevated in BALF from steroid-treated-C-ARDS as well as in NC-ARDS. IL-1β appeared concentrated in BALF, whereas circulating IL-6 and IL-1Ra concentrations were comparable to those in BALF and correlated with severity. TNFα, TNFR1 and CXCL8 however, were significantly higher in NC-ARDS compared to C-ARDS, treated by steroid.ConclusionsIn the lungs of C-ARDS, both caspase-1 activation with a predominant IL-1β/IL-6 signature and IFNγ -associated chemokines are elevated despite steroid treatment. These pathways may be specifically targeted in ARDS to improve response to treatment and to limit alveolar and vascular lung damage.

Tumor development necessitates immune escape through different mechanisms. To counteract these effects, the development of therapies targeting immune checkpoints (ICP) has generated interest as they have produced lasting objective responses in patients with advanced metastatic tumors. However, many tumors do not respond to inhibitors of ICPs, necessitating to further study the underlying mechanisms of exhaustion. VEGFa, a proangiogenic molecule secreted by tumors, was described to participate to tumor immune exhaustion by increasing ICPs, justifying in part the use of an anti-VEGFa mAb, bevacizumab, in patients. However, recent studies from our group have demonstrated that tumors can escape anti-VEGFa therapy through the secretion of soluble CD146 (sCD146). In this study, we show that both VEGFa and sCD146 cooperate to create an immunosuppressive microenvironment by increasing the expression of ICPs. In addition, sCD146 favors protumoral M2-type macrophages and induces the secretion of proinflammatory cytokines. An anti-sCD146 mAb reverses these effects and displays additive effects with the anti-VEGFa antibody to eliminate tumors in a syngeneic murine model grafted with melanoma cells. Combining bevacizumab with mucizumab could thus be of major therapeutic interest to prevent immune escape in malignant melanoma and other CD146-positive tumors.

Supplementary Methods and Materials from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Supplementary Figure Legends 1-3 from Tumor α_vβ₃ Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 auto-antibodies, with subsequent accumulation of circulating ultra-large von Willebrand factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on the surface of dermal human micro-vascular endothelial cell (HMVEC-d), associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)'2 fragments purified from the immunoglobulin G fraction of iTTP plasma mainly induced endothelial cell activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patients' B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small intering RNA, significantly decreased the stimulating effects of iTTP immunoglobulin G. In conclusion, Ca2+-mediated endothelial cell activation constitutes a "second hit" of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

Figure S1 shows Mesenchymal traits of human MDA-B02 and BC-M1 breast cancer cells. Figure S2 shows Quantitative RT-PCR measurements of miR-30 expression levels. Figure S3 shows Quantification of absolute amounts of miR-30s. Figure S4 shows Stable overexpression of miR-30 family members in human MDA-B02 breast cancer cells. Figure S5 shows miR-30s expression in MDA-B02 and BC-M1 breast cancer cells. Figure S6 shows Western blot analyses. Figure S7 shows that CDH11 is a target for repression by miR-30s. Figure S8 shows Interaction of MDA-MB-231 and Hs-578T silenced for CDH11 with MC3T3-E1 osteoblasts. Figure S9 shows ITGB3 and CTGF are direct targets for repression by miR-30s. Figure S10 shows Putative binding sites between miR-30s and the ITGA5 3'UTR. Figure S11 shows effects of miR-30s on MCF-7 tumor cell colonization of the bone marrow ex vivo.

Cardiac fibrosis constitutes irreversible necrosis of the heart muscle as a consequence of different acute (myocardial infarction) or chronic (diabetes, hypertension, …) diseases but also due to genetic alterations or aging. Currently, there is no curative treatment that is able to prevent or attenuate this phenomenon that leads to progressive cardiac dysfunction and life-threatening outcomes. This review summarizes the different targets identified and the new strategies proposed to fight cardiac fibrosis. Future directions, including the use of exosomes or nanoparticles, will also be discussed.

Supplementary Figures 1-3 from Tumor α_vβ₃ Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases

Table S1 shows clinical and pathological characteristics of breast cancer patients. Table S2 shows association of miR-30s expression levels in primary breast tumors with clinicopathological characteristics of breast cancer patients. Table S3 shows prognostic value of miR-30s in breast cancer for RFS. Table S4 shows in silico targets of miR-30 family members.

Supplementary Table 2 from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Supplementary Figure 1 from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Description of (1) retroviral vector constructs, (2) ShRNA design sequences, (3) psiCHECK-2 vector constructs, (4) co-culture of breast tumor cells with osteoblasts, (5) breast cancer cohort of patients, (6) miRNA quantification in primary tumor of breast cancer patients and (7) miRNA profiling of breast cancer cell lines.

Legends to Supplementary Figures S1 to S11.

ObjectiveIL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1β-positive MVs in JIA patients.MethodsMVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs’ ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry.ResultsTHP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients.ConclusionMVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients.

CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

ObjectiveSystemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic‐ and inflammation‐related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc.MethodsWe performed in vitro experiments, including RNA‐Seq and antibody arrays, and in vivo experiments using animal models of bleomycin‐induced SSc and hind limb ischemia.ResultsMultiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM‐10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5‐13‐sCD146 and I10‐sCD146, were identified. Of interest, I5‐13‐sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10‐sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10‐sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5‐13‐sCD146 displayed profibrotic effects through the Wnt‐1/β‐catenin/WISP‐1 pathway.ConclusionVariants of sCD146, and in particular the novel I5‐13‐sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis‐ or fibrosis‐related disorders.

Abstract Rationale Glioblastoma multiforme (GBM) is a primary brain tumor with poor prognosis. The U.S. food and drug administration approved the use of the anti-VEGF antibody bevacizumab in recurrent GBM. However, resistance to this treatment is frequent and fails to enhance the overall survival of patients. In this study, we aimed to identify novel mechanism(s) responsible for bevacizumab-resistance in CD146-positive glioblastoma. Methods The study was performed using sera from GBM patients and human GBM cell lines in culture or xenografted in nude mice. Results We found that an increase in sCD146 concentration in sera of GBM patients after the first cycle of bevacizumab treatment was significantly associated with poor progression free survival and shorter overall survival. Accordingly, in vitro treatment of CD146-positive glioblastoma cells with bevacizumab led to a high sCD146 secretion, inducing cell invasion. These effects were mediated through integrin αvβ3 and were blocked by mucizumab, a novel humanized anti-sCD146 antibody. In vivo, the combination of bevacizumab with mucizumab impeded CD146 + glioblastoma growth and reduced tumor cell dissemination to an extent significantly higher than that observed with bevacizumab alone. Conclusion We propose sCD146 to be 1/ an early biomarker to predict and 2/ a potential target to prevent bevacizumab resistance in patients with glioblastoma.