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 the specific steps outlined in Objectives, this year we concentrated on steps I, II, 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. II. While clinical manifestations of VEDS include patients with haploinsufficiency for COL3A1, so that our experimental studies at 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. To date we were able to prepare 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. In the next step, a 5436 bps fragment containing the mutation of interest and the loxP neo with ApaI/XhoI from pBlueII KS+ mCol3A1mut floxPneo will be cloned in the resulting vector to replace the 3533 bps fragment of the original genomic Col3a1 fragment. In the final step, the resulting vector will be used for generation of the knock in mouse. The phenotype of the produced mouse will be extensively tested for suitability as a model for VEDS. 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 month 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.
|Wang, Mingyi; Zhang, Jing; Telljohann, Richard et al. (2012) Chronic matrix metalloproteinase inhibition retards age-associated arterial proinflammation and increase in blood pressure. Hypertension 60:459-66|
|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|
|Muller, Gerd A; Hansen, Uwe; Xu, Zhi et al. (2012) Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts. FASEB J 26:668-77|
|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|
|Cooper, T K; Zhong, Q; Krawczyk, M et al. (2010) The haploinsufficient Col3a1 mouse as a model for vascular Ehlers-Danlos syndrome. Vet Pathol 47:1028-39|