Notch3 Deficiency Attenuates Pulmonary Fibrosis and Impedes Lung-Function Decline.

Fibroblast activation includes differentiation to myofibroblasts and is a key feature of organ fibrosis. The Notch pathway has been involved in myofibroblast differentiation in several tissues, including the lung. Here, we identify a subset of collagen-expressing cells in the lung that exhibit Notch3 activity at homeostasis. After injury, this activation increases, being found in αSMA-expressing myofibroblasts in the mouse and human fibrotic lung. Although previous studies suggest a contribution of Notch3 in stromal activation, in vivo evidence of the role of Notch3 in lung fibrosis remains unknown. In this study, we examine the effects of Notch3 deletion in pulmonary fibrosis and demonstrate that Notch3-deficient lungs are protected from lung injury with significantly reduced collagen deposition after bleomycin administration. The induction of profibrotic genes is reduced in bleomycin-treated Notch3-knockout lungs that consistently present fewer αSMA-positive myofibroblasts. As a result, the volume of healthy lung tissue is higher and lung function is improved in the absence of Notch3. Using in vitro cultures of lung primary fibroblasts, we confirmed that Notch3 participates in their survival and differentiation. Thus, Notch3 deficiency mitigates the development of lung fibrosis because of its role in mediating fibroblast activation. Our findings reveal a previously unidentified mechanism underlying lung fibrogenesis and provide a potential novel therapeutic approach to target pulmonary fibrosis.

Notch signalling drives synovial fibroblast identity and arthritis pathology.

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.

The frequency of CpG and non-CpG methylation of Notch3 gene promoter determines its expression levels in breast cancer cells.

Notch3 can act as a tumor suppressor in the breast cancer epithelial cells. Unfortunately, Notch3 expression is decreased or lost, especially in triple-negative breast cancer (TNBC) cells, and the reasons remain unclear. Here, we found Notch3 was upregulated in MDA-MB-231 cells with 5-Aza treatment. Two CpG islands were observed in notch3 promoter. Interestingly, bisulfite sequencing exhibited that large amounts of unconverted cytosines were not only followed by guanine, but also adenine, cytosine and thymine, which implied that there simultaneously existed CpG and non-CpG methylation in notch3 promoter. To better analyze the methylation frequency of non-CpG locus, we designed CpG/non-CpG methylation analysis software. The results showed that the methylation frequency of notch3 gene in different breast cancer cell lines was in order T47D, MCF-7, SKBR3, BT-549 and MDA-MB-231. Furthermore, we identified that DNMT3b, DNMT1, DNMT3L, Mecp2 and EZH2 were important regulators of non-CpG locus of notch3 gene. Immunohistochemistry staining revealed a negative correlation between EZH2 and Notch3 from 22 luminal and 26 TNBC cases. In vitro methylation combined luciferase activity assays showed that non-CpG methylation was still crucial cause leading to notch3 transcriptional repression in TNBC. Our findings provide possible explanation for the downregulation or loss of Notch3 expression in TNBC.

Brivanib in combination with Notch3 silencing shows potent activity in tumour models.

BACKGROUND: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results. METHODS: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers. RESULTS: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo. CONCLUSION: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

Evaluation of Notch3 Deficiency in Diabetes-Induced Pericyte Loss in the Retina.

Loss of vascular pericytes has long been associated with the onset of diabetic retinopathy; however, mechanisms contributing to pericyte dropout are not understood. Notch3 has been implicated in pericyte stability and survival, and linked to vascular integrity. Notch3 mutant mice exhibit progressive loss of retinal pericytes. Given that diabetic retinopathy is associated with pericyte loss, we sought to determine whether perturbation of Notch3 signaling contributes to diabetes-induced pericyte dropout and capillary degeneration. We utilized a pericyte-expressed LacZ transgene (XlacZ4) to examine pericyte loss in retinas of a type I diabetic mouse model (Ins2Akita) and Notch3-deficient mice. Notch3 null animals showed a dramatic loss of the LacZ marker by 8 weeks of age, while Ins2Akita diabetic and Notch3 heterozygous mice exhibited a much slower and subtler loss of LacZ. Although combined Notch3 heterozygosity in Ins2Akita diabetic animals did not show further deficits, the trypsin digest method revealed that Notch3 haploinsufficiency increased the formation of acellular capillaries in diabetic mice. Our data further indicate that Notch signaling is blunted in diabetic retinas and in cells exposed to hyperglycemia. These results are the first to demonstrate an association between Notch3 signaling, pericyte loss, and diabetic retinopathy.

Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone.

In the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout the mammal's lifetime. The balance between quiescence and active cell division among NSCs is crucial in producing appropriate numbers of neurons while maintaining the stem cell pool for a long period. The Notch signaling pathway plays a central role in both maintaining quiescent NSCs (qNSCs) and promoting cell division of active NSCs (aNSCs), although no one knows how this pathway regulates these apparently opposite functions. Notch1 has been shown to promote proliferation of aNSCs without affecting qNSCs in the adult mouse subependymal zone (SEZ). In this study, we found that Notch3 is expressed to a higher extent in qNSCs than in aNSCs while Notch1 is preferentially expressed in aNSCs and transit-amplifying progenitors in the adult mouse SEZ. Furthermore, Notch3 is selectively expressed in the lateral and ventral walls of the SEZ. Knockdown of Notch3 in the lateral wall of the adult SEZ increased the division of NSCs. Moreover, deletion of the Notch3 gene resulted in significant reduction of qNSCs specifically in the lateral and ventral walls, compared with the medial and dorsal walls, of the lateral ventricles. Notch3 deletion also reduced the number of qNSCs activated after antimitotic cytosine ß-D-arabinofuranoside (Ara-C) treatment. Importantly, Notch3 deletion preferentially reduced specific subtypes of newborn neurons in the olfactory bulb derived from the lateral walls of the SEZ. These results indicate that Notch isoforms differentially control the quiescent and proliferative steps of adult SEZ NSCs in a domain-specific manner.SIGNIFICANCE STATEMENT In the adult mammalian brain, the subependymal zone (SEZ) of the lateral ventricles is the largest neurogenic niche, where neural stem cells (NSCs) generate neurons. In this study, we found that Notch3 plays an important role in the maintenance of quiescent NSCs (qNSCs), while Notch1 has been reported to act as a regulator of actively cycling NSCs. Furthermore, we found that Notch3 is specifically expressed in qNSCs located in the lateral and ventral walls of the lateral ventricles and regulates neuronal production of NSCs in a region-specific manner. Our results indicate that Notch3, by maintaining the quiescence of a subpopulation of NSCs, confers a region-specific heterogeneity among NSCs in the adult SEZ.

Notch3 deficiency impairs coronary microvascular maturation and reduces cardiac recovery after myocardial ischemia.

RATIONALE: Vascular maturation plays an important role in wound repair post-myocardial infarction (MI). The Notch3 is critical for pericyte recruitment and vascular maturation during embryonic development. OBJECTIVE: This study is to test whether Notch3 deficiency impairs vascular maturation and blunts cardiac functional recovery post-MI. APPROACH AND RESULTS: Wild type (WT) and Notch3 knockout (Notch3KO) mice were subjected to MI by the ligation of left anterior descending coronary artery (LAD). Cardiac function and coronary blood flow reserve (CFR) were measured by echocardiography. The expression of angiogenic growth factor, pericyte/capillary coverage and arteriolar formation were analyzed. Loss of Notch3 in mice resulted in a significant reduction of pericytes and small arterioles. Notch3 KO mice had impaired pericyte/capillary coverage and CFR compared to WT mice. Notch3 KO mice were more prone to ischemic injury with larger infarcted size and higher rates of mortality. The expression of CXCR-4 and VEGF/Ang-1 was significantly decreased in Notch3 KO mice. Notch3 KO mice also had few NG2+/Sca1+ and NG2+/c-kit+ progenitor cells in the ischemic area and exhibited worse cardiac function recovery at 2weeks after MI. These were accompanied by a significant reduction of pericyte/capillary coverage and arteriolar maturation. Furthermore, Notch3 KO mice subjected to MI had increased intracellular adhesion molecule-2 (ICAM-2) expression and CD11b+ macrophage infiltration into ischemic areas compared to that of WT mice. CONCLUSION: Notch3 mutation impairs recovery of cardiac function post-MI by the mechanisms involving the pre-existing coronary microvascular dysfunction conditions, and impairment of pericyte/progenitor cell recruitment and microvascular maturation.