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The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3-/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3-/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfbret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.
Assuntos
Cardiomegalia , Hipertrofia Ventricular Esquerda , Animais , Camundongos , Becaplermina , Metabolismo dos Lipídeos , Miócitos Cardíacos , Proteínas Proto-Oncogênicas c-sisRESUMO
All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.
Assuntos
Linhagem da Célula , Sistema Nervoso Central , Macrófagos , Sistema Nervoso Central/imunologia , Feminino , Humanos , Imunidade Inata , Macrófagos/citologia , Microglia , Gravidez , Saco VitelinoRESUMO
OBJECTIVE: To conduct a clinical study of a family with neurologic symptoms and findings carrying a novel NOTCH3 mutation and to analyze the molecular consequences of the mutation. METHODS: We analyzed a family with complex neurologic symptoms by MRI and neurologic examinations. Exome sequencing of the NOTCH3 locus was conducted, and whole-genome sequencing was performed to identify COL4A1, COL4A2, and HTRA1 mutations. Cell lines expressing the normal or NOTCH3A1604T receptors were analyzed to assess proteolytic processing, cell morphology, receptor routing, and receptor signaling. RESULTS: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease (SVD) and caused by mutations in the NOTCH3 gene. Most CADASIL mutations alter the number of cysteine residues in the extracellular domain of the NOTCH3 receptor, but in this article, we describe a family in which some members carry a novel cysteine-sparing NOTCH3 mutation (c.4810 G>A, p.Ala1604Thr). Two of 3 siblings heterozygous for the NOTCH3A1604T mutation presented with migraine and white matter lesions (WMLs), the latter of a type related to but distinct from what is normally observed in CADASIL. Two other members instead carried a novel COL4A1 missense mutation (c.4795 G>A; p.(Ala1599Thr)). The NOTCH3A1604T receptor was aberrantly processed, showed reduced presence at the cell surface, and less efficiently activated Notch downstream target genes. CONCLUSIONS: We identify a family with migraine and WML in which some members carry a cysteine-sparing hypomorphic NOTCH3 mutation. Although a causal relationship is not established, we believe that the observations contribute to the discussion on dysregulated Notch signaling in cerebral SVDs.
RESUMO
Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between the NOTCH and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, where an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.
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Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.
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PURPOSE: Systematic family-centered cancer care is needed. We conducted a randomized controlled trial of family therapy, delivered to families identified by screening to be at risk from dysfunctional relationships when one of their relatives has advanced cancer. PATIENTS AND METHODS: Eligible patients with advanced cancer and their family members screened above the cut-off on the Family Relationships Index. After screening 1,488 patients or relatives at Memorial Sloan Kettering Cancer Center or three related community hospice programs, 620 patients (42%) were recruited, which represented 170 families. Families were stratified by three levels of family dysfunction (low communicating, low involvement, and high conflict) and randomly assigned to one of three arms: standard care or 6 or 10 sessions of a manualized family intervention. Primary outcomes were the Complicated Grief Inventory-Abbreviated (CGI) and Beck Depression Inventory-II (BDI-II). Generalized estimating equations allowed for clustered data in an intention-to-treat analysis. RESULTS: On the CGI, a significant treatment effect (Wald χ(2) = 6.88; df = 2; P = .032) and treatment by family-type interaction was found (Wald χ(2) = 20.64; df = 4; P < .001), and better outcomes resulted from 10 sessions compared with standard care for low-communicating and high-conflict groups compared with low-involvement families. Low-communicating families improved by 6 months of bereavement. In the standard care arm, 15.5% of the bereaved developed a prolonged grief disorder at 13 months of bereavement compared with 3.3% of those who received 10 sessions of intervention (Wald χ(2) = 8.31; df = 2; P =.048). No significant treatment effects were found on the BDI-II. CONCLUSION: Family-focused therapy delivered to high-risk families during palliative care and continued into bereavement reduced the severity of complicated grief and the development of prolonged grief disorder.
Assuntos
Luto , Terapia Familiar , Neoplasias/terapia , Adulto , Comunicação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/psicologiaRESUMO
BACKGROUND: Supporting the family-as-a-whole presents challenges in palliative care, although family meetings are increasingly used in routine practice. The Family Focused Grief Therapy (FFGT) Model guides clinicians in using a range of intervention strategies. AIM: To examine the therapists' techniques used in assessing 'at risk' families in palliative care to better illuminate what helps and what remains challenging. METHOD: Recorded sessions 1 and 2 were coded using the FFGT fidelity coding measure, with its glossary of definitions. Inter-rater reliability between three coders was satisfactory at 88%. Frequencies of strategy utilization were computed, with extraction of examples of both successful and problematic approaches. SETTING/PARTICIPANTS: From within a larger study of family therapy during palliative care at a comprehensive cancer center, the first two sessions (n = 144) delivered to 74 families (299 individuals) by 32 therapists were coded and analyzed. RESULTS: Therapists readily explored the story of illness and families' ways of coping (97%) and assessed communication and cohesiveness in the majority. Exploration of relational patterns occurred in 89% of sessions, use of a genogram in 80%, understanding members' roles in 65% and family values and beliefs in 62%. Less use was made of summaries (39%), family mottos (34%), exploration of family conflict (35%) and the formalization of a comprehensive family treatment plan (20%). CONCLUSIONS: Challenges exist in therapy with difficult families. Therapy in the home brings special issues. Therapists can apply most of the interventions prescribed by the FFGT model.