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Notch3 Deficiency Impairs Vascular Smooth Muscle Cell Contractility and Glymphatic Function in the Brain

ABSTRACT: While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cell (vSMC) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3-deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3-/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3-/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3-/- . MRI and behavioral assessments were performed in the Notch3-/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.

ORGANISM(S): Mus musculus

PROVIDER: GSE204803 | GEO | 2024/01/17

REPOSITORIES: GEO

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Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

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Notch3 Deficiency Impairs Vascular Smooth Muscle Cell Contractility and Glymphatic Function in the Brain

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