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Competing Interests: Competing interests: L.K. has no competing interest. K.H. is co-founder and shareholder of Sequantrix GmbH, R.K. is a founder, shareholder, and board member of Sequantrix GmbH; is a member of the scientific advisory board of Hybridize Therapeutics; has received honoraria for advisory boards and talks from Bayer, Chugai, Pfizer, Roche, Genentech, Eli Lilly, AMGEN, Novo Nordisk and GlaxoSmithKline; and has research funding from Travere Therapeutics, Galapagos, Novo Nordisk and AskBio.

Microvascular inflammation (MVI) in kidney transplant biopsies is mainly associated with antibody-mediated rejection (AMR), sparking debate within the Banff Classification of Renal Allograft Pathology regarding its exclusivity. This study reviewed the literature on MVI in T cell-mediated rejection (TCMR) and analyzed MVI in our transplant population. We searched English publications in MEDLINE, Embase, Web of Science, Cochrane, and Google Scholar until June 2024, focusing on glomerulitis (g), peritubular capillaritis (ptc), or MVI in kidney transplant biopsies classified as TCMR. Additionally, we examined g, ptc, and MVI in 69 patients with AMR, TCMR, and no rejection. Our search yielded 541 citations, with 10 studies included, covering 810 TCMR and 156 AMR biopsies. The studies showed g, ptc, and MVI were present in TCMR but were less prevalent and severe than in AMR. In our cohort, AMR had significantly higher g, ptc, and MVI scores compared to aTCMR and ATN, however, aTCMR also displayed MVI. These findings confirm that MVI occurs in aTCMR and should not be exclusively linked to AMR. These findings highlight the need to further explore MVI's significance in TCMR and investigate the inflammatory composition. This could refine the Banff Classification, improving Classification accuracy of kidney transplant pathology assessments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Varol, Wagenmakers, Hoeft, Callemeyn, Bodewes, Bramer, Stubbs, Kramann, Naesens and Clahsen-Van Groningen.)

Single cell RNA sequencing has provided unprecedented insights into the molecular cues and cellular heterogeneity underlying human disease. However, the high costs and complexity of single cell methods remain a major obstacle for generating large-scale human cohorts. Here, we compare current state-of-the-art single cell multiplexing technologies, and provide a widely applicable demultiplexing method, SoupLadle, that enables simple, yet robust high-throughput multiplexing leveraging genetic variability of patients., Competing Interests: Competing interests: R.K. is a founder, shareholder and board member of Sequantrix GmbH, a member of the scientific advisory board of Hybridize Therapeutics, and has received honoraria for advisory boards and talks from Bayer, Chugai, Pfizer, Roche, Genentech, Lilly, and GSK and has received research funding from Travere Therapeutics, Galapagos, Novo Nordisk and Ask Bio. S.H. reports funding from Novo Nordisk and Ask Bio. K.H. and S.H. are co-founders of Sequantrix GmbH. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Ferroptosis is a regulated cell death modality triggered by iron-dependent lipid peroxidation. Ferroptosis plays a causal role in the pathophysiology of various diseases, making it a promising therapeutic target. Unlike all other cell death modalities dependent on distinct signaling cues, ferroptosis occurs when cellular antioxidative defense mechanisms fail to suppress the oxidative destruction of cellular membranes, eventually leading to cell membrane rupture. Physiologically, only two such surveillance systems are known to efficiently prevent the lipid peroxidation chain reaction by reducing (phospho)lipid hydroperoxides to their corresponding alcohols or by reducing radicals in phospholipid bilayers, thus maintaining the integrity of lipid membranes. Mechanistically, these two systems are linked to the reducing capacity of glutathione peroxidase 4 (GPX4) by consuming glutathione (GSH) on one hand and ferroptosis suppressor protein 1 (FSP1, formerly AIFM2) on the other. Notably, the importance of ferroptosis suppression in physiological contexts has been linked to a particular vulnerability of renal tissue. In fact, early work has shown that mice genetically lacking Gpx4 rapidly succumb to acute renal failure with pathohistological features of acute tubular necrosis. Promising research attempting to implicate ferroptosis in various renal disease entities, particularly those with proximal tubular involvement, has generated a wealth of knowledge with widespread potential for clinical translation. Here, we provide a brief overview of the involvement of ferroptosis in nephrology. Our goal is to introduce this expanding field for clinically versed nephrologists in the hope of spurring future efforts to prevent ferroptosis in the pathophysiological processes of the kidney., (© The Author(s) 2024. Published by Oxford University Press on behalf of the ERA.)

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.

Introduction: The tobacco endgame, policies aiming to end the commercial tobacco epidemic, requires sustained public support, including among youth. We assessed endgame support among California (USA) adolescents, including their reasons and associated participant and policy-specific factors., Methods: Teens, Nicotine and Tobacco Project online surveys (n=4827) and focus groups were conducted in 2021 and 2022 among California residents aged 12-17 years. Cross-sectional survey participants were asked their agreement level with eight policy statements related to tobacco and/or cannabis sales restrictions, use in public places and use in multiunit housing. Ordered logistic regression modelled level of agreement according to respondent characteristics, behaviours and statement content. Qualitative data were collected through focus groups (n=51 participants), which were analysed to provide insight into support for different policies., Results: Most survey participants agreed or strongly agreed with tobacco product sales restrictions (72%-75%, depending on the policy), bans on use in public spaces (76%-82%) and smoke-free (79%) and vape-free (74%) apartment buildings. Support was stronger among younger, female, Asian and tobacco non-using participants and for policies directed at 'tobacco' (vs 'vapes' or cannabis), at flavoured tobacco (compared with all tobacco), and when statements featured 'should end' (vs 'not allowed'). Focus group participants who were supportive viewed policies as protecting children from harmful products, while those less supportive cited concerns about limiting adults' freedoms and unintended consequences., Conclusions: Most participants supported strong tobacco control policies. Public communication that promotes broader endgame benefits besides protecting youth and accelerates industry denormalisation may counter youth concerns and further bolster their support., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24 + epithelial subpopulation. Long-term-cultured CD24 + cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Myocardial infarction is a leading cause of death worldwide 1 . Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality 2 . Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling., (© 2022. The Author(s).)

Background & Aims: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia., Methods: We analyzed the intestine-specific villin-Cre DSP (DSP ΔIEC ) and the combined intestine-specific DSG2/DSP ΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay., Results: The intestine of DSP ΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSP ΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSP ΔIEC /keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch., Conclusions: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Childhood malnutrition has been a longstanding crisis in Mumbai, India. Despite national IYCF (Infant Young Child Feeding) guidelines to promote best practices for infant/toddler feeding, nearly one-third of children under age five are stunted or underweight. To improve child nutrition, interventions should address the cultural, social, and environmental influences on infant feeding practices. This study is an in-depth qualitative assessment of family barriers and facilitators to implementing recommended nutrition practices in two Mumbai slum communities, within the context of an existing nutrition education-based intervention by a local non-governmental non-profit organization., Methods: The population was purposively sampled to represent a variety of household demographics. Data were collected through 33 in-depth semi-structured interviews with caregivers (mothers and paternal grandmothers) of children age 0-2 years. Transcripts were translated and transcribed, and analyzed using qualitative analysis procedures and software., Results: A complex set of barriers and facilitators influence mothers'/caregivers' infant-toddler feeding practices. Most infants were fed complementary foods and non-nutritious processed snacks, counter to IYCF recommendations. Key barriers included: lack of nutrition knowledge and experience, receiving conflicting messages from different sources, limited social support, and poor self-efficacy for maternal decision-making. Key facilitators included: professional nutrition guidance, personal self-efficacy and empowerment, and family support. Interventions to improve child nutrition should address mothers'/caregivers' key barriers and facilitators to recommended infant-toddler feeding practices., Conclusions: Nutrition interventions should prioritize standard messaging across healthcare providers, engage all family members, target prevention of early introduction of sugary and non-nutritious processed foods, and strengthen maternal self-efficacy for following IYCF recommended guidelines.

Objective: After a first episode of lower extremity deep venous thrombosis, post-thrombotic syndrome (PTS) develops in 20% to 50% of patients despite adequate anticoagulation. Symptoms of PTS can vary from leg swelling to venous ulceration with disabling venous claudication. It significantly affects the patient's quality of life and has considerable socioeconomic consequences. This review gives an update on diagnosis and current treatment strategies in patients with PTS due to chronic venous obstruction, in particular regarding the role of endovenous procedures., Methods: This review article is based on a selective literature search in PubMed and the Cochrane Library. The terms "postthrombotic syndrome," "post-thrombotic syndrome," "chronic venous obstruction," "venous outflow obstruction," and "venous stent" were used as keywords. Selected publications addressed the diagnosis of and therapy for PTS. Acute deep venous thrombosis, thrombolysis, case reports, complications as a result of caval vein filters, animal experiments, PTS of the upper extremity, and PTS in children were excluded., Results: In addition to conservative treatment of PTS, the following invasive procedures are also available: open surgical reconstructions, hybrid procedures, and endovenous recanalization of the occluded iliocaval venous tract with stent angioplasty. Since introduction of dedicated venous stents in 2012, technical success, patency rates, and improvement in quality of life have been at least as good as results of open surgical reconstruction if not better., Conclusions: First-line treatment should be conservative therapy. In case of therapy-resistant PTS with poor quality of life, the possibility of an invasive treatment should be evaluated. All invasive procedures are recommended with low levels of evidence. Therefore, deciding on an invasive treatment and type of procedure should be made individually. Because PTS is rarely a threat to life or limb, a minimally invasive treatment is preferred. Therefore, endovenous recanalization appears to be appropriate as the therapy of choice. In patients with involvement of the femoral confluence, endophlebectomy of the common femoral vein in addition to venous recanalization is inevitable to ensure an adequate inflow into the recanalized venous tract. It also secures a sufficient drainage of blood from the peripheral venous system. Because this hybrid procedure is burdened with a significantly higher risk of complications, strict criteria must be fulfilled to legitimize the indication for this procedure. For the best possible results to be achieved, the following perioperative and postoperative management must be considered: therapeutic anticoagulation, early mobilization, compression therapy, and systematic follow-up with duplex ultrasound., (Copyright © 2019 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Background: In vascular surgery, novel synthetic prosthesis materials for patch-angioplasties, interpositions, bypasses and shunts are continuously under development and optimization. The characteristics of an ideal vascular prosthesis would display long-term patency, biocompatibility, durability, low porosity, lack of stich hole bleeding, ease of handling, kink resistance, infection resistance and reasonable costs. The aim of this study was to establish and report a reliable sheep model including potential pitfalls where those parameters could be analyzed. Before surgery, sheep were acclimatized for 4-8 weeks, during which parasite infections were treated and blood and serum parameters monitored. Twenty-four sheep underwent surgery, and carotid patch-angioplasties (n = 12), graft interpositions (n = 6) or arteriovenous prosthetic shunts (n = 6) were implanted. Half of the animals in each group were sacrificed after 2 weeks and the other half after 8 weeks. The implants were analyzed for patency, endothelialization, thrombogenicity and biocompatibility by clinical observation, blood flow measurement and pathological and histopathological (H&E, EvG) as well as immunohistochemical (Ki67, CD31) evaluations., Results: Health monitoring of the sheep revealed a parasitic burden with endoparasites in all animals. Some animals showed thereby infestations in the bile duct causing fibrotic cholangitis with calcifications in the liver. In addition, sarcosporidia were detected in histopathological specimen of the heart in all animals. Parasitic burden correlated with blood counts and serum bilirubin levels. Both were significantly reduced by albendazole treatment within the acclimatization time. Patches, interposition grafts, and straight shunts were successfully implanted bilaterally in all animals. The total average operation time was 136 ± 21 min. Most animals (23/24) showed good patency rates and general condition after implantation. Pathological and histopathological/immunohistochemical analyses were suitable to determine thrombogenicity, endothelialization, cellular/fibroblastic proliferation, biocompatibility, inflammatory cell infiltration, and thickness of neointima in the prosthesis material., Conclusions: We have developed a suitable experimental protocol with standardized and successful anesthesia- and surgical-procedures for patch-angioplasty, graft interposition, and arteriovenous prosthetic shunts. This sheep model allows testing of new prosthetic materials for biocompatibility, thrombogenicity, and endothelialization.

Fibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells, including mesenchymal stem cells, macrophages, and fibrocytes, to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium, we confirm that the proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single-cell RNA sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes, and express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms.

Background: Perioperative and critically ill patients are often exposed to iron (in the form of parenteral-iron administration or blood transfusion) and inflammatory stimuli, but the effects of iron loading on the inflammatory response are unclear. Recent data suggest that mitochondrial reactive oxygen species have an important role in the innate immune response and that increased mitochondrial reactive oxygen species production is a result of dysfunctional mitochondria. We tested the hypothesis that increased intracellular iron potentiates lipopolysaccharide-induced inflammation by increasing mitochondrial reactive oxygen species levels., Methods: Murine macrophage cells were incubated with iron and then stimulated with lipopolysaccharide. C57BL/6 wild-type mice were intraperitoneally injected with iron and then with lipopolysaccharide. Markers of inflammation and mitochondrial superoxide production were examined. Mitochondrial homeostasis (the balance between mitochondrial biogenesis and destruction) was assessed, as were mitochondrial mass and the proportion of nonfunctional to total mitochondria., Results: Iron loading of mice and cells potentiated the inflammatory response to lipopolysaccharide. Iron loading increased mitochondrial superoxide production. Treatment with MitoTEMPO, a mitochondria-specific antioxidant, blunted the proinflammatory effects of iron loading. Iron loading increased mitochondrial mass in cells treated with lipopolysaccharide and increased the proportion of nonfunctional mitochondria. Iron loading also altered mitochondrial homeostasis to favor increased production of mitochondria., Conclusions: Acute iron loading potentiates the inflammatory response to lipopolysaccharide, at least in part by disrupting mitochondrial homeostasis and increasing the production of mitochondrial superoxide. Improved understanding of iron homeostasis in the context of acute inflammation may yield innovative therapeutic approaches in perioperative and critically ill patients.

Objectives: To determine the effectiveness of the Contra Caries Oral Health Education Program (CCOHEP) for improving low-income, Spanish-speaking parents' oral health knowledge and behaviors for their young children. Mexican American children in the United States suffer disproportionately high prevalence and severity of early childhood caries, yet few evaluated, theory-based behavioral interventions exist for this population. CCOHEP is a theory-based curriculum consisting of four 2-h interactive classes designed for and by Spanish speakers and led by designated community health educators (promotoras). Topics included children's oral hygiene, caries etiology, dental procedures, nutrition, child behavior management, and parent skill-building activities., Methods: Low-income Spanish-speaking parents/caregivers of children aged 0-5 years were recruited through community services in an agricultural city in California. Survey questions from the Oral Health Basic Research Facts Questionnaire measuring oral health-related behaviors and knowledge were verbally administered before, immediately after, and 3 months after attendance at CCOHEP. Five questions measured aspects of parental toothbrushing for their children (frequency, using fluoridated toothpaste, brushing before bed, not drinking or eating after nighttime brushing, adult assistance), three questions measured other oral health behaviors, and 16 questions measured oral health-related knowledge. Analyses of within-person changes between pre- and post-tests and again between post-test and 3-month follow-up consisted of McNemar's test for binary outcomes and sign tests for ordinal outcomes., Results: Overall, 105 caregivers participated in CCOHEP (n = 105 pretest, n = 95 post-test, n = 79 second post-test). At baseline, all parents self-reported doing at least one aspect of toothbrushing correctly, but only 13% reported performing all five aspects according to professional guidelines. At post-test, 44% of parents reported completing all aspects of toothbrushing according to professional guidelines (P < .001). Statistically significant improvements were seen in 4 aspects of toothbrushing (P ≤ .008) between pretest and post-test (all but adult assistance). The second post-test showed 3 of these improvements were maintained, while adult assistance and the other reported behaviors improved (P ≤ .008). Between pretest and post-test, checking child's teeth monthly and frequency of sweet drinks consumption improved (P ≤ .008), while frequency of eating sweet foods did not change. Knowledge was high at baseline (mean 12.8 of 16), but 6 knowledge items improved significantly between pretest and post-test. Improvements were maintained at second post-test., Conclusions: Contra Caries Oral Health Education Program improved low-income Spanish-speaking parents' oral hygiene knowledge and self-reported behaviors for their young children, and change was sustained 3 months after the end of the intervention. Future, more rigorous evaluation of the intervention is recommended., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Matrix Gla protein (MGP) is reported to inhibit bone morphogenetic protein (BMP) signal transduction. MGP deficiency is associated with medial calcification of the arterial wall, in a process that involves both osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and mesenchymal transition of endothelial cells (EndMT). In this study, we investigated the contribution of BMP signal transduction to the medial calcification that develops in MGP-deficient mice., Approach and Results: MGP-deficient mice (MGP(-/-)) were treated with one of two BMP signaling inhibitors, LDN-193189 or ALK3-Fc, beginning one day after birth. Aortic calcification was assessed in 28-day-old mice by measuring the uptake of a fluorescent bisphosphonate probe and by staining tissue sections with Alizarin red. Aortic calcification was 80% less in MGP(-/-) mice treated with LDN-193189 or ALK3-Fc compared with vehicle-treated control animals (P<0.001 for both). LDN-193189-treated MGP(-/-) mice survived longer than vehicle-treated MGP(-/-) mice. Levels of phosphorylated Smad1/5 and Id1 mRNA (markers of BMP signaling) did not differ in the aortas from MGP(-/-) and wild-type mice. Markers of EndMT and osteogenesis were increased in MGP(-/-) aortas, an effect that was prevented by LDN-193189. Calcification of isolated VSMCs was also inhibited by LDN-193189., Conclusions: Inhibition of BMP signaling leads to reduced vascular calcification and improved survival in MGP(-/-) mice. The EndMT and osteogenic transdifferentiation associated with MGP deficiency is dependent upon BMP signaling. These results suggest that BMP signal transduction has critical roles in the development of vascular calcification in MGP-deficient mice.