Vacular type of Ehlers-Danlos Syndrome: experimental models and treatment

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The broad objective of this program is to perform preclinical experimentation on the mouse genetic models of VEDS to test the efficacy of different therapeutic modalities to attenuate the vascular fragility and, thus, to prevent or to reduce the risk of vascular complications. With respect to specific steps outlined in Objectives, this year we concentrated on steps I, III, and V. Characterization of existing genetic model of VEDS I. The haploinsufficiency for one COL3A1 allele is one of the genotypes resulting in VEDS. Homozygous Col3a1 mice, the only currently available and described model of VEDS, cannot be used for experiments due to extremely high prenatal mortality. We hypothesized, that heterozygous Col3a1 mice, originally described as phenotypically normal, can serve as an experimental model of haploinsufficiency. We compared aortas and colons of 2, 5, 9, 14 and 21 month old (+/-) and (+/+) Col3a1 mice, males and females in vivo (high frequency sonography and pressure-volume analyses) and ex vivo (histology and biomechanical properties). In all ages the aorta in +/- mice had a lower ex vivo rupture pressure, than in wild type mice. Histological evaluation of aortas of +/- mice demonstrated lower content of collagen (Sirius red staining) and lesions among 100% of male and 50% of female mice older than 5-mo of age, revealing elastin fragmentation, spindle cell proliferation, inflammation, and reactive fibrosis. The colon of +/- animals had higher compliance and lower maximal (rupture) pressure (higher fragility) in 9-21 month old animals. This was associated with a lower collagen III content detected by quantitative RT-PCR. Thus, the +/- Col3A1 mouse could serve as an experimental model for the VEDS. III. One approach to a targeted treatment of VEDS is to eliminate the mutated form of the COL3A1 gene and thus to transform the more severe form of VEDS (mutation) to the less severe haploinsufficient type of VEDS. We attempted to knock down the expression of the mRNA product of the mutated allele by RNA interference with siRNAs using fibroblasts from a VEDS patient heterozygous for COL3A1 allele encoding a glycine substitution, Gly85 -> Val (G85V), which is the most common class of mutation causing VEDS. We systematically identified small interfering RNAs (siRNAs) that discriminate between the wild-type and the mutant COL3A1 allele by a luciferase reporter gene assay, total of 19. The three best siRNAs were further tested in primary fibroblasts from a normal donor and a VEDS patient. The best discriminative siRNA resulted in 90% silencing of the mutated allele. The reduced amount of the mutated allele of COL3A1 mRNA abolished the unfolded protein response of the endoplasmatic reticulum. Conclusion: Allele specific siRNAs treatment was able to reduce the negative effects of mutated proteins by reducing the unfolded protein response of the endoplasmatic reticulum. Thus the use of allele-specific RNAi may be an option to reduce the severity of VEDS after improving the delivery method of siRNA. This treatment should be tested specifically in tissue culture from patients, but not in the artificial testing system. V. We hypothesized that treatment with broad spectrum metall proteinase (MMP) inhibitor, doxycycline would prevent or attenuate the development of aortic lesions in (-/+) COL3A1 mice. Six months old females (-/+) and wild type mice were treated for 3 months with the broad spectrum MMP inhibitor doxycycline (25 mg/kg per day). At the end of the treatment, under general inhalation narcosis (2% of isoflurane in oxygen) the aortas were surgically stressed: for 30 sec the blood flow was stopped by pressing the cotton-tip applicator at the level of renal arteries to elevate the blood pressure above stoppage. One week after intervention aortas were harvested and processed. The number of lesions was counted in serial sections every 2 mm across aortas. The average number of lesions in untreated wild type mice was 0.90.32. In untreated (-/+) mice the average number of lesions was elevated to 2.30.40 (p<0.05); the average number of lesions in (-/+) mice treated with doxycycline was 1.10.50 - significantly (p<0.05) lower than in untreated animals and similar to wild type mice. The result suggests that doxycycline merits clinical testing as a treatment for VEDS. Development of means to suppress specific mutation in tissue culture experiments. The experiments conducted during this year led to a patent application, which is currently being filed, and thus cannot be disclosed at this time.