Homozygous plasminogen activator inhibitor-i (PAI-1 )-deficient (PAI-1 /-I) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-i was demonstrated by Southern blot analysis. A 3.0-kb PAI-i-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-i +1+) but not from PAI-1i--mice. Plasma PAI-i levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63±2 ng/ml, 30±10 ng/ml, and undetectable (< 2 ng/ ml) in PAl-i ++, heterozygous (PA-i +/-) and PAMl-/-mice, respectively (mean±SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1 +/+ but not of PAl-i--mice. SDS-gel electrophoresis of plasma incubated with '25I-labeled recombinant human tissue-type plasminogen activator revealed an 115,000-M, component with plasma from endotoxin-stimulated (0.5 mg/kg) PA-i +/+ but not from PAM--/-mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAM-i-mice were viable, produced similar sizes of litters as PA-+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities. (J. Clin. Invest. 1993. 92:2746-2755

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ABSTRACT

Objective-Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease. Methods and Results-Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3 Arg170Cys mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3Arg170Cys mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis. The main symptoms are migraine with aura, psychiatric disturbances, and recurrent subcortical ischemic strokes causing motor disabilities, cognitive decline, dementia, and premature death. 2,3 The accompanying arteriopathy is characterized by the progressive degeneration of arterial smooth muscle cells (SMCs), deposition of electron-dense granular osmiophilic material (GOM) within the basal lamina of the SMCs and pericytes, and accumulation of the Notch3 extracellular cleavage product at the SMC membrane. 2-7 Although the arteriopathy is systemic, 8 the symptoms are mostly neurological. Brain pathology involves microbleeds, loss of myelin or axons, fibrillar gliosis, and lacunar infarcts in the white and deep gray matter. 2,3,9 CADASIL is caused by mutations in the transmembrane receptor Notch3, which is predominantly expressed in adult arterial SMCs. 10 -13 Pathogenic mutations stereotypically affect the number of cysteine residues in the epidermal growth factor-like repeats of the extracellular domain (ECD). [13][14][15] Thus far, more than 180 mutations are known, nearly 60% of which cluster in exon 4. 3,[13][14][15][16] In vitro and in vivo studies have highlighted a role of Notch3 in artery maturation, responses to vascular injury or ischemia, and regulation of SMC proliferation and apoptosis. [17][18][19][20][21][22][23][24][25] How the various Notch3 mutations effectuate arteriopathy and CADASIL disease development remains enigmatic.Previous approaches to generating a model of CADASIL include knock-in mice carrying the counterpart Arg141Cys CADASIL mutation (Arg142Cys in mouse) in the endogenous Notch3 gene, which failed, however, to develop any phenotype at all. 26 On the other hand, transgenic overexpression in mice of human or rat Notch3 carrying archetypal Notch3 mutations (Arg90Cys human Notch3, 27-30 Cys428Ser human Notch3, 31 Arg169Cys rat Notch3, 32 Cys455Arg and Arg1031Cys human Notch3 33 ) met with more success, with all models displaying the CADASIL arteriopathy. However, Here, we report a novel mutant mouse carrying an Arg170Cys knock-in mutation in the...