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 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. III. Marfan syndrome (MFS) is another systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in Marfan syndrome patients is associated with aortic dilatations and valvular insufficiency;however, it remains unclear whether myocardial alterations are prompted by a valvular pathology or if they develop independently. We studied the age-dependent progression of cardiac remodeling and left ventricular dysfunction in the mouse model of MFS known as Fbn1039G+/- mouse (Marfan HT mouse). Hearts of 2-4 month old and older (6-14 mo) HT and wild type Marfan mice were examined by echocardiography, pressure-volume loop analyses, and histologically. Marfan HT mice of 2-4 months demonstrated a hypertrophic cardiac remodeling accompanied by a decline of predominantly diastolic function and increased TGF-b canonical (p SMAD2/3) and non-canonica l (pERK and MAPK38) signaling. Valvular function remained largely unaffected in this age group. Hypertrophic cardiac remodeling among older HT mice developed into two distinctly different phenotypes associated with either dilated or reduced (constricted) LV chamber. Diastolic dysfunction in the older HT mice was combined with significant systolic impairment. The aortic valve regurgitation was registered in 20% of constricted group and 60% of dilated, while mitral insufficiency was observed in 40% of constricted group and 100% of dilated, suggesting that LV dilatation was a major contributor to a mitral valve failure. In Marfan HT mice, extracellular matrix abnormalities and phosphorylation of SMAD2/3 were not associated with the increase of interstitial fibrosis. In the mouse model fibrillin-1 haploinsufficiency results in the early onset of hypertrophic cardiac remodeling and dysfunction that is not associated with functional valvular abnormalities. With age, cardiac remodeling progressed into two distinct hypertrophic phenotypes with diminishing cardiac function characterized either by left ventricular concentric hypertrophy or dilatation with increased frequency of valvular backflow in dilated group.
