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

该计划的主要目的是对VEDS的小鼠遗传模型进行临床前实验，以测试不同治疗方式减轻血管脆性的有效性，从而预防或降低血管并发症的风险。关于目标中概述的具体步骤，今年我们集中注意步骤一、二和五。 VEDS现有遗传模型的表征 I.一个COL 3A 1等位基因的单倍不足是导致VEDS的基因型之一。 纯合子Col 3a 1小鼠，目前唯一可用和描述的VEDS模型，不能用于实验，由于极高的产前死亡率。我们假设，杂合子Col 3a 1小鼠，最初被描述为表型正常，可以作为单倍不足的实验模型。 我们比较了2、5、9、14和21个月大（+/-）和（+/+）Col 3a 1小鼠（雄性和雌性）体内（高频超声和压力-容积分析）和离体（组织学和生物力学特性）的结肠和结肠。 在所有年龄段中，+/-小鼠的主动脉离体破裂压低于野生型小鼠。对+/-小鼠的结缔组织的组织学评价表明，在100%的雄性和50%的雌性小鼠中，5月龄以上的胶原蛋白含量（天狼星红染色）和病变较低，显示弹性蛋白断裂、梭形细胞增殖、炎症和反应性纤维化。在9-21月龄动物中，+/-动物的结肠具有较高的顺应性和较低的最大（破裂）压力（较高的脆性）。这与定量RT-PCR检测到的较低的III型胶原含量有关。 因此，+/-Col 3A 1小鼠可以作为VEDS的实验模型。 二.虽然VEDS的临床表现包括Col 3A 1单倍不足的患者，因此我们对单倍不足小鼠模型的实验研究具有临床相关性，但这种特定遗传问题的患病率仅代表受影响人群的一小部分。大多数受影响的患者代表不同类型的突变;最典型的是编码甘氨酸取代的COL 3A 1等位基因的异源性，Gly 85->瓦尔（G85 V），这是导致VEDS的最常见的突变类型。G85 V突变是由COL 3A 1 cDNA编码区第755位鸟嘌呤变为胸苷引起的。因此，我们着手设计一种反映上述突变的小鼠。 迄今为止，我们能够制备2种含有Southern探针的载体，一种含有Col 3a 1基因组序列的12.8kb片段（从内含子1到内含子14）的载体和一种含有目的突变（外显子10中的G854 T）和loxP neo的载体。在下一步中，将含有目的突变和来自pBlueII KS+ mCol 3A 1 mut pneo的具有ApaI/XhoI的loxP neo的5436 bp片段克隆到所得载体中，以取代原始基因组Col 3a 1片段的3533 bp片段。 在最后一步中，所得载体将用于在小鼠中产生敲除。将广泛检测所生产小鼠的表型作为VEDS模型的适用性。 诉我们假设用广谱金属蛋白酶（MMP）抑制剂强力霉素治疗可以预防或减轻（-/+）COL 3A 1小鼠主动脉病变的发展。 用广谱MMP抑制剂多西环素（25 mg/kg/天）处理6个月大的雌性（-/+）和野生型小鼠3个月。在处理结束时，在全身吸入麻醉（2%异氟烷的氧气溶液）下，通过手术对肾动脉施加压力：通过在肾动脉水平按压棉尖施用器使血压升高至停止水平以上，使血流停止30秒。干预后一周，收获并加工了果蝇。在连续切片中每隔2 mm计数病变数量。未处理的野生型小鼠中病变的平均数量为0.90.32。在未处理的（-/+）小鼠中，病变的平均数量升高至2.30.40（p<0.05）;用多西环素处理的（-/+）小鼠中病变的平均数量为1.10.50 -显著（p<0.05）低于未处理的动物并且与野生型小鼠相似。结果表明，强力霉素值得临床试验作为治疗VEDS。

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.