Activation of the RAS pathway through uncommon BRAF mutations in mucinous pancreatic cysts without KRAS mutation.

Diagnostic testing of pancreatic cyst fluid obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has traditionally utilized elevated carcinoembryonic antigen (CEA) (≥192 ng/ml) and cytomorphologic examination to differentiate premalignant mucinous from benign pancreatic cystic lesions (PCLs). Molecular testing for KRAS/GNAS mutations has been shown to improve accuracy of detecting mucinous PCLs. Using a targeted next-generation sequencing (NGS) panel, we assess the status of PCL-associated mutations to improve understanding of the key diagnostic variables. Molecular analysis of cyst fluid was performed on 108 PCLs that had concurrent CEA and/or cytological analysis. A 48-gene NGS assay was utilized, which included genes commonly mutated in mucinous PCLs such as GNAS, KRAS, CDKN2A, and TP53. KRAS and/or GNAS mutations were seen in 59 of 68 (86.8%) cases with multimodality diagnosis of a mucinous PCL. Among 31 patients where surgical histopathology was available, the sensitivity, specificity, and diagnostic accuracy of NGS for the diagnosis of mucinous PCL was 88.5%, 100%, and 90.3%, respectively. Cytology with mucinous/atypical findings were found in only 29 of 62 cases (46.8%), with fluid CEA elevated in 33 of 58 cases (56.9%). Multiple KRAS mutations at different variant allele frequencies were seen in seven cases favoring multiclonal patterns, with six of them showing at least two separate PCLs by imaging. Among the 6 of 10 cases with GNAS + /KRAS- results, uncommon, non-V600E exon 11/15 hotspot BRAF mutations were identified. The expected high degree of accuracy of NGS detection of KRAS and/or GNAS mutations for mucinous-PCLs, as compared with CEA and cytological examination, was demonstrated. Multiple KRAS mutations correlated with multifocal cysts demonstrated by radiology. In IPMNs that lacked KRAS mutations, the concurring BRAF mutations with GNAS mutations supports an alternate mechanism of activation in the Ras pathway.

Essential role of stress hormone signaling in cardiomyocytes for the prevention of heart disease.

Heart failure is a leading cause of death in humans, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in cardiovascular health and disease is poorly understood. To determine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of the glucocorticoid receptor (GR). These mice are born at the expected Mendelian ratio, but die prematurely from spontaneous cardiovascular disease. By 3 mo of age, mice deficient in cardiomyocyte GR display a marked reduction in left ventricular systolic function, as evidenced by decreases in ejection fraction and fractional shortening. Heart weight and left ventricular mass are elevated, and histology revealed cardiac hypertrophy without fibrosis. Removal of endogenous glucocorticoids and mineralocorticoids neither augmented nor lessened the hypertrophic response. Global gene expression analysis of knockout hearts before pathology onset revealed aberrant regulation of a large cohort of genes associated with cardiovascular disease as well as unique disease genes associated with inflammatory processes. Genes important for maintaining cardiac contractility, repressing cardiac hypertrophy, promoting cardiomyocyte survival, and inhibiting inflammation had decreased expression in the GR-deficient hearts. These findings demonstrate that a deficiency in cardiomyocyte glucocorticoid signaling leads to spontaneous cardiac hypertrophy, heart failure, and death, revealing an obligate role for GR in maintaining normal cardiovascular function. Moreover, our findings suggest that selective activation of cardiomyocyte GR may represent an approach for the prevention of heart disease.

Size effects and electronic properties of zinc-doped boron clusters Zn B n (n = 1-15).

CONTEXT: To gain a deeper understanding of zinc-doped boron clusters, theoretical calculations were performed to investigate the size effects and electronic properties of zinc-doped boron clusters. The study of the electronic properties, spectral characteristics, and geometric structures of Zn B n (n = 1-15) is of great significance in the fields of semiconductor materials science, material detection, and improving catalytic efficiency. The results indicate that Zn B n (n = 1-15) clusters predominantly exhibit planar or quasi-planar structures, with the Zn atom positioned in the outer regions of the B n framework. The second stable structure of Zn B 3 is a three-dimensional configuration, indicating that the structures of zinc-doped boron clusters begin to convert from the planar or quasi-planar structures to the 3D configurations. The second low-energy structure of Zn B 15 is a novel configuration. Relative stability analyses show that the Zn B 12 has better chemical stability than other clusters with a HOMO-LUMO gap of 2.79 eV. Electric charge analysis shows that part electrons on zinc atoms are transferred to boron atoms, and electrons prefer to cluster near the B n framework. According to the electron localization function, it gets harder to localize electrons as the equivalent face value drops, and it's challenging to see covalent bond formation between zinc and boron atoms. The spectrograms of Zn B n (n = 1-15) exhibit distinct properties and notable spectral features, which can be used as a theoretical basis for the identification and confirmation of boron clusters doped with single-atom transition metals. METHODS: The calculations were performed using the ABCluster global search technique combined with density functional theory (DFT) methods. The selected low-energy structures were subjected to geometric optimization and frequency calculations at the PBE0/6-311 + G(d) level to ensure structural stability and eliminate any imaginary frequencies. To acquire more precise relative energies, we performed single-point energies calculations for the low-lying isomers of Zn B n (n = 1-15) at the CCSD(T)/6-311 + G(d)//PBE0/6-311 + G(d) level of theory. All calculations were performed using Gaussian 09 software. To facilitate analysis, we utilized software tools such as Multiwfn, and VMD.

Bone morphogenetic protein endothelial cell precursor-derived regulator regulates retinal angiogenesis in vivo in a mouse model of oxygen-induced retinopathy.

OBJECTIVE: Bone morphogenetic proteins (BMPs) are potently proangiogenic; however, the mechanisms underlying the regulation of vessel development by BMPs are not fully understood. To assess the significance of BMP endothelial cell precursor-derived regulator (BMPER) in blood vessel formation in vivo, we investigated its role in retinal angiogenesis. METHODS AND RESULTS: In a model of oxygen-induced retinopathy, Bmper mRNA expression and protein levels are downregulated, correlating with the initiation of Sma and Mad related protein phosphorylation in endothelial cells. Moreover, Bmper haploinsufficiency results in an increased rate of retinal revascularization, with retinas from Bmper+/- mice displaying increased numbers of branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Furthermore, although Bmper haploinsufficiency does not alter Bmp expression, it does lead to an increase in BMP signaling, as evidenced by increased phosphorylated Sma and Mad related protein levels in endothelial cells and increased expression of known BMP target genes. CONCLUSIONS: These observations provide compelling evidence that BMPER is important in the regulation of BMP signaling and revascularization in the hypoxic retina. These bring forth the possibility of novel therapeutic approaches for pathological angiogenesis based on manipulation of BMP signaling.

An unusual Aspergillus, herpes and strongyloides triple infection in a patient with chronic pulmonary disease: A case report with literature review.

We report a case of a 75 year old non-known cancer or organ transplant male with an unusual concurrent triple infection of Aspergillus, strongyloides stercoralis and herpes simplex virus in a bronchoalveolar lavage. He presented to an outside hospital with worsening respiratory distress and an open tracheostomy was performed due to concern he would not extubate. Following tracheostomy, there was concern for a possible esophageal perforation. A bronchoalveolar lavage (BAL) were performed and Strongyloides, herpes viral cytopathic changes and aspergillus microorganisms were identified. The patient subsequently expired following discharge.

Targeting HER2 heterogeneity in breast cancer.

The identification of Human epidermal growth factor receptor 2 (HER2) as a target in breast cancer and the subsequent development of HER2-targeted therapies has revolutionized the treatment of patients with HER2-positive breast cancer. However, there is an increasing awareness of how frequently tumors have low or heterogeneous expression of HER2. It is now recognized that this impacts the degree of benefit from HER2-targeted therapies. With the advent of novel and more potent antibody drug conjugates, targeting HER2 in traditional HER2-negative tumors with "HER2-low" expression is becoming possible. It is essential to refine the nomenclature around HER2 expression to enable clinicians to optimize treatment for patients across the HER2 expression spectrum in breast cancer. HER2 heterogeneity can be detected by conventional IHC, gene expression profiling or other methods and numerous studies have documented the correlation between the presence of HER2 heterogeneity and shorter disease-free survival (DFS) and overall survival (OS). Validation of techniques to identify HER2 heterogeneity in the clinic and concurrent development of agents to effectively treat tumors with non-uniform HER2 expression is needed.

Interobserver agreement in programmed cell death-ligand 1 immunohistochemistry scoring in nonsmall cell lung carcinoma cytologic specimens.

BACKGROUND: The evaluation of PD-L1 expression in nonsmall cell lung carcinoma (NSCLC) is becoming increasingly important given the effectiveness of PD-L1 inhibitors. Although cytologic specimens have been shown to be compatible with surgical specimens to evaluate PD-L1 immunohistochemistry (IHC), evidence of the reproducibility of PD-L1 in cytologic specimens is lacking. The aim of this study is to evaluate interobserver agreement in PD-L1 IHC in cytologic specimens. METHODS: PD-L1 IHC was performed on 86 NSCLC cytology specimens using Dako PD-L1 IHC 22C3 pharmDx. The digitally scanned whole slide images (WSI) were read by five pathologists. Each case was given a Tumor Proportion Score (TPS) and the results were compared between the observers. The interobserver concordance was assessed using 1% and 50% as cutoffs. RESULTS: TPSs were highly correlated among observers (Spearman correlation coefficient, 0.86-0.94). Using greater than 1% as a cutoff, interobserver agreement measured by Fleiss Kappa was 0.74 for all pathologists and Cohen's Kappa coefficient ranged from 0.49 to 0.83, consistent with moderate to substantial agreement. With a cutoff of greater than 50%, Fleiss Kappa was 0.79 for all pathologists and the kappa values ranged from 0.63 to 0.90, consistent with substantial to almost perfect agreement. Several pitfalls were identified by reviewing discordant cases, including staining in macrophages, stromal cells, and intratumoral heterogeneity. CONCLUSION: Our data suggest that TPS of PD-L1 IHC on cytology specimens is reproducible, with a better agreement when using 50% as the cutoff value. However, special attention is required when the TPS is near the 1% cutoff.

Lipid laden macrophages and electronic cigarettes in healthy adults.

BACKGROUND: An outbreak of E-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) with significant morbidity and mortality was reported in 2019. While most patients with EVALI report vaping tetrahydrocannabinol (THC) oils contaminated with vitamin E acetate, a subset report only vaping with nicotine-containing electronic cigarettes (e-cigs). Whether or not e-cigs cause EVALI, the outbreak highlights the need for identifying long term health effects of e-cigs. EVALI pathology includes alveolar damage, pneumonitis and/or organizing pneumonia, often with lipid-laden macrophages (LLM). We assessed LLM in the lungs of healthy smokers, e-cig users, and never-smokers as a potential marker of e-cig toxicity and EVALI. METHODS: A cross-sectional study using bronchoscopy was conducted in healthy smokers, e-cig users, and never-smokers (n = 64). LLM, inflammatory cell counts, and cytokines were determined in bronchial alveolar fluids (BAL). E-cig users included both never-smokers and former light smokers. FINDINGS: High LLM was found in the lungs of almost all smokers and half of the e-cig users, but not those of never-smokers. LLM were not related to THC exposure or smoking history. LLM were significantly associated with inflammatory cytokines IL-4 and IL-10 in e-cig users, but not smoking-related cytokines. INTERPRETATION: This is the first report of lung LLM comparing apparently healthy smokers, e-cig users, and never-smokers. LLM are not a specific marker for EVALI given the frequent positivity in smokers; whether LLMs are a marker of lung inflammation in some e-cig users requires further study. FUNDING: The National Cancer Institute, the National Heart, Lung, and Blood Institute, the Food and Drug Administration Center for Tobacco Products, the National Center For Advancing Translational Sciences, and Pelotonia Intramural Research Funds.

Biphasic chest wall synovial sarcoma with epithelial pleural effusion: a diagnostic challenge.

Synovial sarcoma (SS) is a rare malignancy that most commonly involves the extremities and large joints. We describe a 67-year-old woman who presented with shortness of breath and flu-like symptoms, and a chest wall mass. On resection of the mass biphasic morphology of SS was noted, as well as confirmatory immunostains including TLE1 and bcl2. An SS18/SSX2 fusion transcript was detected by reverse transcriptase-DNA amplification. A year later, following chemotherapy, the patient developed a right-sided pleural effusion. Cytological examination of the fluid showed an epithelial population forming clusters and groups. TLE1 was positive, as well as fluorescent in situ hybridization analysis for the SS18/SSX2 fusion transcript. SS can be a challenging diagnosis in fluid-filled cavities, when the epithelial component predominates and its original biphasic quality is not seen. We discuss the diagnostic challenges of monophasic and biphasic SS, and updates to ancillary testing.

PRDM6 is enriched in vascular precursors during development and inhibits endothelial cell proliferation, survival, and differentiation.

The mechanisms that regulate the differentiation program of multipotential stem cells remain poorly understood. In order to define the cues that delineate endothelial commitment from precursors, we screened for candidate regulatory genes in differentiating mouse embryoid bodies. We found that the PR/SET domain protein, PRDM6, is enriched in flk1(+) hematovascular precursor cells using a microarray-based approach. As determined by 5' RACE, full-length PRDM6 protein contains a PR domain and four Krüppel-like zinc fingers. In situ hybridization in mouse embryos demonstrates staining of the primitive streak, allantois, heart, outflow tract, paraaortic splanchnopleura (P-Sp)/aorto-gonadal-mesonephric (AGM) region and yolk sac, all sites known to be enriched in vascular precursor cells. PRDM6 is also detected in embryonic and adult-derived endothelial cell lines. PRDM6 is co-localized with histone H4 and methylates H4-K20 (but not H3) in vitro and in vivo, which is consistent with the known participation of PR domains in histone methyltransferase activity. Overexpression of PRDM6 in mouse embryonic endothelial cells induces apoptosis by activating caspase-3 and inducing G1 arrest. PRDM6 inhibits cell proliferation as determined by BrdU incorporation in endothelial cells, but not in rat aortic smooth muscle cells. Overexpression of PRDM6 also results in reduced tube formation in cultured endothelial cells grown in Matrigel. Taken together, our data indicate that PRDM6 is expressed by vascular precursors, has differential effects in endothelial cells and smooth muscle cells, and may play a role in vascular precursor differentiation and survival by modulating local chromatin-remodeling activity within hematovascular subpopulations during development.

Gene expression profile signatures indicate a role for Wnt signaling in endothelial commitment from embryonic stem cells.

We have used global gene expression analysis to establish a comprehensive list of candidate genes in the developing vasculature during embryonic (ES) cell differentiation in vitro. A large set of genes, including growth factors, cell surface molecules, transcriptional factors, and members of several signal transduction pathways that are known to be involved in vasculogenesis or angiogenesis, were found to have expression patterns as expected. Some unknown or functionally uncharacterized genes were differentially regulated in flk1+ cells compared with flk1- cells, suggesting possible roles for these genes in vascular commitment. Particularly, multiple components of the Wnt signaling pathway were differentially regulated in flk1+ cells, including Wnt proteins, their receptors, downstream transcriptional factors, and other components belonging to this pathway. Activation of the Wnt signal was able to expand vascular progenitor populations whereas suppression of Wnt activity reduced flk1+ populations. Suppression of Wnt signaling also inhibited the formation of matured vascular capillary-like structures during late stages of embryoid body differentiation. These data indicate a requisite and ongoing role for Wnt activity during vascular development, and the gene expression profiles identify candidate components of this pathway that participate in vascular cell differentiation.

Giant cell tumor of temporomandibular joint presenting as a parotid tumor: Challenges in the accurate subclassification of giant cell tumors in an unusual location.

Fine needle aspiration is frequently used as the initial diagnostic procedure in the work-up of head and neck lesions, including soft tissue masses and salivary gland neoplasms. Giant cell tumors (GCTs), both osseous and extraosseous, are benign tumors that occur, albeit rarely, in the head and neck region. Extraosseous GCTs may be further classified based on their tissue of origin and specific anatomic location. Regardless of location, giant cell tumors are morphologically similar and share cytologic and histologic diagnostic criteria. Evaluation of imaging is therefore essential to the correct classification of these tumors. Accurate diagnosis is crucial since the clinical behavior and treatment is significantly different among the subtypes of GCTs. The case presented herein illustrates the diagnostic dilemma between two uncommon entities in an unusual site: GCT of parotid gland and tenosynovial GCT.

BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.

The development of endothelial cell precursors is essential for vasculogenesis. We screened for differentially expressed transcripts in endothelial cell precursors in developing mouse embryoid bodies. We cloned a complete cDNA encoding a protein that contains an amino-terminal signal peptide, five cysteine-rich domains, a von Willebrand D domain, and a trypsin inhibitor domain. We termed this protein BMPER (bone morphogenetic protein [BMP]-binding endothelial cell precursor-derived regulator). BMPER is specifically expressed in flk-1-positive cells and parallels the time course of flk-1 induction in these cells. In situ hybridization in mouse embryos demonstrates dorsal midline staining and staining of the aorto-gonadal-mesonephric region, which is known to host vascular precursor cells. BMPER is a secreted protein that directly interacts with BMP2, BMP4, and BMP6 and antagonizes BMP4-dependent Smad5 activation. In Xenopus embryos, ventral injection of BMPER mRNA results in axis duplication and downregulation of the expression of Xvent-1 (downstream target of Smad signaling). In an embryoid body differentiation assay, BMP4-dependent differentiation of endothelial cells in embryoid bodies is also antagonized by BMPER. Taken together, our data indicate that BMPER is a novel BMP-binding protein that is expressed by endothelial cell precursors, has BMP-antagonizing activity, and may play a role in endothelial cell differentiation by modulating local BMP activity.

BMPER regulates cardiomyocyte size and vessel density in vivo.

BACKGROUND: BMPER, an orthologue of Drosophila melanogaster Crossveinless-2, is a secreted factor that regulates bone morphogenetic protein activity in endothelial cell precursors and during early cardiomyocyte differentiation. Although previously described in the heart, the role of BMPER in cardiac development and function remain unknown. METHODS: BMPER-deficient hearts were phenotyped histologically and functionally using echocardiography and Doppler analysis. Since BMPER -/- mice die perinatally, adult BMPER +/- mice were challenged to pressure-overload-induced cardiac hypertrophy and hindlimb ischemia to determine changes in angiogenesis and regulation of cardiomyocyte size. RESULTS: We identify for the first time the cardiac phenotype associated with BMPER haploinsufficiency. BMPER messenger RNA and protein are present in the heart during cardiac development through at least E14.5 but is lost by E18.5. BMPER +/- ventricles are thinner and less compact than sibling wild-type hearts. In the adult, BMPER +/- hearts present with decreased anterior and posterior wall thickness, decreased cardiomyocyte size and an increase in cardiac vessel density. Despite these changes, BMPER +/- mice respond to pressure-overload-induced cardiac hypertrophy challenge largely to the same extent as wild-type mice. CONCLUSION: BMPER appears to play a role in regulating both vessel density and cardiac development in vivo; however, BMPER haploinsufficiency does not result in marked effects on cardiac function or adaptation to pressure overload hypertrophy.

Dual role for glucocorticoids in cardiomyocyte hypertrophy and apoptosis.

Glucocorticoids and their synthetic derivatives are known to alter cardiac function in vivo; however, the nature of these effects and whether glucocorticoids act directly on cardiomyocytes are poorly understood. To explore the role of glucocorticoid signaling in the heart, we used rat embryonic H9C2 cardiomyocytes and primary cardiomyocytes as model systems. Dexamethasone (100 nm) treatment of cardiomyocytes caused a significant increase in cell size and up-regulated the expression of cardiac hypertrophic markers, including atrial natriuretic factor, ß-myosin heavy chain, and skeletal muscle α-actin. In contrast, serum deprivation and TNFα exposure triggered cardiomyocyte apoptosis, and these apoptotic effects were inhibited by dexamethasone. Both the hypertrophic and anti-apoptotic actions of glucocorticoids were abolished by the glucocorticoid receptor (GR) antagonist RU486 and by short hairpin RNA-mediated GR depletion. Blocking the activity of the mineralocorticoid receptor had no effect on these glucocorticoid-dependent cardiomyocyte responses. Aldosterone (1 µm) activation of GR also promoted cardiomyocyte hypertrophy and cell survival. To elucidate the mechanism of the dual glucocorticoid actions, a genome-wide microarray was performed on H9C2 cardiomyocytes treated with vehicle or dexamethasone in the absence or presence of serum. Serum dramatically influenced the transcriptome regulated by GR, revealing potential glucocorticoid signaling mediators in both cardiomyocyte hypertrophy and apoptosis. These studies reveal a direct and dynamic role for glucocorticoids and GR signaling in the modulation of cardiomyocyte function.

Glucocorticoid signaling in cardiac disease.

As major mediators of stress regulation, glucocorticoids have an essential role in maintaining cardiovascular homeostasis under both physiological and pathological conditions. The release of glucocorticoids into the peripheral circulation is adjusted by the hypothalamic-pituitary-adrenal axis in response to various pathological challenges such as sepsis, starvation, and psychological stress. Clinically, dysregulation of the glucocorticoid-mediated signaling as a result of either excess ligand or receptor hypersensitivity is connected with the progression of unfavorable cardiovascular events such as cardiac hypertrophy, atherosclerosis, and coronary artery disease. The direct effects of glucocorticoids on cardiac tissues are mediated by two steroid receptors, the glucocorticoid receptor and mineralocorticoid receptor, which are both expressed by cardiomyocytes. Although each receptor has some shared responses to glucocorticoids, each receptor also has unique effects on cardiac functions. Elucidating the selective actions of each receptor is critical for determining the proper pharmaceutical targets in cardiovascular diseases.

A concentration-dependent endocytic trap and sink mechanism converts Bmper from an activator to an inhibitor of Bmp signaling.

Bmper, which is orthologous to Drosophila melanogaster crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators.

Gene expression profiles identify a role for cyclooxygenase 2-dependent prostanoid generation in BMP6-induced angiogenic responses.

The bone morphogenetic protein (BMP) family of proteins participates in regulation of angiogenesis in physiologic and pathologic conditions. To investigate the molecular mechanisms that contribute to BMP-dependent angiogenic signaling, we performed gene expression profiling of BMP6-treated mouse endothelial cells. We detected 77 mRNAs that were differentially regulated after BMP6 stimulation. Of these, cyclooxygenase 2 (Cox2) was among the most highly up-regulated by BMP stimulation, suggesting a role for Cox2 as a downstream regulator of BMP-induced angiogenesis. Up-regulation of Cox2 by BMP6 was detected at both mRNA and protein levels in endothelial cells, and BMP6 increased production of prostaglandins in a Cox2-dependent fashion. BMP6 up-regulated Cox2 at the transcriptional level through upstream SMAD-binding sites in the Cox2 promoter. Pharmacologic inhibition of Cox2, but not Cox1, blocked BMP6-induced endothelial cell proliferation, migration, and network assembly. BMP6-dependent microvessel outgrowth was markedly attenuated in aortic rings from Cox2-/- mice or after pharmacologic inhibition of Cox2 in aortas from wild-type mice. These results support a necessary role for Cox2 in mediating proangiogenic activities of BMP6. These data indicate that Cox2 may serve as a unifying component downstream from disparate pathways to modulate angiogenic responses in diseases in which neovascularization plays an underlying pathophysiologic role.

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors.

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.