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Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

ABSTRACT: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation. However, the underlying cellular and molecular mechanisms remain unidentified. Here, we generated non-integrative induced pluripotent stem cells (iPSCs) from fibroblasts from a CADASIL patient harboring a heterozygous NOTCH3 mutation (c.3226C>T, p.R1076C). Vascular smooth muscle cells (VSMCs) differentiated from CADASIL-specific iPSCs showed transcriptional changes and disease-associated cellular dysfunction, including activation of the NF-kB signaling pathway, cytoskeleton disorganization, and excessive cell proliferation. In comparison, these abnormalities were not observed in vascular endothelial cells (VECs) derived from the patient’s iPSCs. Importantly, the abnormal upregulation of NF-kB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor, providing a potential therapeutic strategy for CADASIL. Overall, using this iPSC-based disease model, our study identified clues for studying the disease mechanisms of CADASIL and developing treatment strategies for this disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124500 | GEO | 2018/12/31

REPOSITORIES: GEO

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Project description: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.

Project description:The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease, accounting for ~10-15% of disease among non-African populations. The ~60kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Genetic studies implicate the 9p21.3 locus and other risk genes to effects in the vascular wall. Here, we use genome editing to delete the entire risk on non-risk haplotype from the genomes of human iPSCs and perform genomewide transcriptional profiling along the timecourse of their differentiation into vascular smooth muscle cells (VSMCs). These studies identify a network of ~3000 genes governed by the risk haplotype in VSMCs that predict deficits in cell division, adhesion and contraction, which we confirmufunctionally. Remarkably, deleting the risk haplotype reverts VSMCs to resemble the non-risk VSMCs, suggesting that the risk region drives a cell state transition. transcriptionally and functionally. . Deleting the risk haplotype reverts these cells to reverted to the non-risk of iPSCs we show that the non-risk haplotype has little effect on locus we produce iPSCs from risk and non-risk individuals, delete each haplotype using genome editing and generate vascular smooth muscle cells (VSMCs). We show that risk VSMCs exhibit aberrant adhesion and contraction, concomitant with dramatically altered global transcriptional changes that are enriched in previously identified cardiovascular disease genes and pathways. Unexpectedly, deleting the risk haplotype rescues VSMC transcriptional identity and function, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This studies shows that the risk haplotype dominantly predisposes VSMCs to adopt perturbed phenotypes associated with cardiovascular disease and establishes haplotype-edited iPSCs as powerful tools for functionally annotating human-specific variation in non-coding genomic regions.

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Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

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