The broad objective of this program is to develop mouse genetic models of VEDS, characterize the phenotype of these models, and to perform preclinical experimentation on these 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. I. Characterization of existing genetic model of VEDS 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 investigated the heterozygous Col3a1 knockout mouse as a model for this disease. We found that the collagen content in the abdominal aorta of heterozygotes was reduced, and functional testing revealed diminishing wall strength of the aorta in these mice. Although mice did not present any life-threatening clinical signs or gross lesions of vascular subtype Ehlers-Danlos syndrome type IV, thorough histological examination of the aorta of heterozygous mice revealed the presence of a spectrum of lesions similar to those observed in human patients. Lesions increased in number and severity with age (0/5 0% in 2-month-old males vs 9/9 100% in 14-month-old males, P <0.05) and were more common in male than female mice (23/26 88.5% vs 14/30 46.7% in 9- to 21-month-old animals, P <0.05). We concluded that haploinsufficiency for Col3a1 in mice recapitulates features of vascular Ehlers-Danlos syndrome in humans and can be used as an experimental model. II. Develop a new, more specific model of VEDS While clinical manifestations of VEDS include patients with haploinsufficiency for COL3A1, and our experimental studies of the mouse model of haploinsifficiency are clinically relevant, the prevalence of this particular genetic problem represents only a small portion of the affected population. Most affected patients represent different kinds of mutations;most typically a heterozygousity for COL3A1 allele encoding a Glycine substitution, Gly85 ->Val (G85V), which is the most common class of mutation causing VEDS. The G85V mutation is caused by the exchange from a Guanine to a Thymidine at position 755 in the coding region of COL3A1 cDNA. Thus, we set out to engineer a mouse that reflected the above mutation. We prepared 2 vectors containing southern probes, a vector containing a 12.8 kb fragment of the genomic sequence of Col3a1 (from intron 1 to intron 14) and a vector containing the mutation of interest (G854T in exon 10) and loxP neo. A 5436 bps fragment containing the mutation of interest and the loxP neo with ApaI/XhoI from pBlueII KS+ mCol3A1mut floxPneo had also been cloned in the resulting vector to replace the 3533 bps fragment of the original genomic Col3a1 fragment. Currently, the resulting vector is being used for generation of the knockout in mouse, which phenotype will be extensively tested for suitability as a model for VEDS. III. Develop means to suppress specific mutation in tissue culture experiments The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant negative mutations in the procollagen type III (COL3A1) gene and has a normal amount of mutated collagen III. Patients that are haploinsufficient for COL3A1 have a reduced amount of non-mutated COL3A1, and seem to exhibit less severe symptoms and subsequent complications. One approach to a targeted treatment of vEDS is the elimination of the mutated form of the COL3A1 gene to transform the more severe phenotype to the less severe haploinsufficient type. We tested the effectiveness of allele specific RNA interference (RNAi) to eliminate the mutated phenotype in fibroblasts. Small interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a vEDS patient. The best discriminative siRNA with mutation at position 10 resulted in more than 90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the vEDS patient revealed structurally abnormal fibrils. After siRNA treatment collagen fibrils were not distinguishable from fibroblasts of normal donors. In these fibrils collagens types I and III were present. Allele specific siRNAs reduced negative effects of mutated COL3A1 proteins by stimulating reconstitution of normal collagen fibrils. Thus the use of allele-specific RNAi may be one option for future personalized therapy to reduce the severity of vEDS. IV and V. Develop specific surgical or pharmacological manipulation that would unmask and allow measuring hidden vascular fragility;Test different therapeutic prophylactic interventions to reduce vascular fragility. We established that surgical manipulation of aortas in heterozygous COL3A1 mice consisting of repeated interruption of the aortic blood flow by pressing and releasing the aorta resulted in 3-fold increase in the number of lesion in aortic wall. It is postulated that weakness of the arterial wall in vEDS (haploinsufficiency) is associated with degradation (without proper restoration) of the wall ECM. We hypothesize that the use of matrix metalloproteinase (MMP) inhibitors will stop or slow down this process. Tetracyclines are mostly known as antibiotics, but recent work has shown that doxycycline, and other derivates of tetracycline, are also potent broad-spectrum MMP inhibitors. The only MMP inhibitor of this type so far approved as a therapeutic drug is the oral tetracycline, doxycycline, for the treatment of peridontitis and rosacea. It is used in below anti-bacterial doses. It was also shown that oral application of doxycycline elevated the norepinephrine-induced collagen accumulation in the heart. Heterozygous Col3a1 knockout mouse (HT) has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether change of the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. Following 3-mo treatment with doxycycline or placebo, 9-mo old HT or wild-type mice (WT) mice were subjected to a surgical manipulation involving a mechanical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT one week after intervention (cumulative score 4.5+-0.87 vs. 1.3+-0.34 in wild-type, p<0.001) was fully prevented in the doxycycline treated group (1.1+-0.56, p<0.001). The untreated HT showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta, whereas, in doxycycline treated animals there was normalization to the levels observed in WT. Results proved that doxycycline treatment inhibits activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS. In another experiment we hypothesize that treatment with doxycycline started early in life will prevent the development of spontaneous lesions in arterial wall of haploinsuffucien Col3a1 mice. Doxycycline was added to food of heterozygous mice started at the time of weaning (3 weeks of age). Results indicated that in the 9mo old haploinsufficient mice the number of spontaneous aortic lesions was reduced to the level observed in the wild type control. The reduction of lesions was associated with increase of collagen content in aortic wall.
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|Tae, Hyun-Jin; Marshall, Shannon; Zhang, Jing et al. (2012) Chronic treatment with a broad-spectrum metalloproteinase inhibitor, doxycycline, prevents the development of spontaneous aortic lesions in a mouse model of vascular Ehlers-Danlos syndrome. J Pharmacol Exp Ther 343:246-51|
|Briest, Wilfried; Cooper, Timothy K; Tae, Hyun-Jin et al. (2011) Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome. J Pharmacol Exp Ther 337:621-7|
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