Extracellular Matrix and Stress Substrates: the Role of Prolidase

我们希望建立一种反映人类遗传性代谢紊乱表型的脯氨酸酶缺乏症动物模型。这种人类疾病的特点是伤口愈合不良，腿部溃疡，骨骼异常和智力低下。然而，在人类身上的这些发现并不一致。家系已被描述，其中具有相同生化表型的兄弟姐妹可能具有不同的临床表现。为了进行这些研究，我们与蒙大拿州大瀑布的麦克劳克林医学研究所的特蕾莎·冈恩博士建立了合作关系。她描述了一种在毛色上有异常的小鼠品系(dal/dal)。她培育了一群纯合子的Dal/Dal小鼠，然后发现编码脯氨酸酶的多肽基因有4个碱基对的缺失。这些动物的表型包括肥厚型心肌病。我们建立了这些小鼠的群体，由于背景品系不确定，我们获得了杂合子，用C3H培育了Dal/Dal，并获得了F2动物，用红细胞脯氨酸酶测定WT和Dal/Dal。随后，研究人员将小猫伴侣作为研究的对照。执行了两个方案。首先，对伤口进行5 mm的穿孔活组织检查。对照创面用赋形剂处理，而实验创面用不同剂型的一氧化氮制剂(ProliNO)处理，该制剂释放出脯氨酸和一氧化氮。我们发现使用的普罗利诺水平是有毒的，因为它抑制了伤口愈合。重要的是，WT和Dal/Dal小鼠之间没有明显的差异。我们尝试的另一种方案是两阶段皮肤肿瘤发生模型。同样，我们发现WT和DAQL/DAL在乳头状瘤的产生方面没有差异。因此，我们不得不得出结论，在这些条件下，脯氨酸酶活性不是限制因素。尽管伤口愈合方面的结果令人失望，但从我们在意大利的合作者那里传来了一些令人兴奋的消息。帕维亚大学的安东内拉·帕维亚博士在Dal/Dal小鼠身上发现了一些惊人的骨骼异常。降低了股骨长度，减少了胫骨干骺端和骨干端的皮质骨和骨小梁面积，明显影响了骨骼的发育。Forlino博士正在进行这些研究，将其作为患有脯氨酸酶缺乏症的人类骨骼异常的模型。

We desired an animal model for prolidase deficiency which reflected the phenotype in humans with this inherited metabolic disorder. The human disorder is characterized by poor wound healing with ulcers on the lower extremities, bony abnormalities and mental retardation. These findings in humans, however, are not consistent. Pedigrees have been described in which siblings with identical biochemical phenotype may have variable clinical manifestations. To pursue these studies, we established a collaboration with Dr. Teresa Gunn of the McLaughlin Medical Research Institute in Great Falls, Montana. She has described a mouse strain (dal/dal) which have abnormalities in coat color. Breeding a colony of homozygous dal/dal mice, she then identified a 4-bp deletion in the pepd gene encoding prolidase. The phenotype in these animals included hypertrophic cardiomyopathy. We established a colony of these mice and since the backgrouind strain was indeterminate, we obtained heterozygotes breeding dal/dal with C3H and obtained F2 animals which were tested for red cell prolidase to determine WT and dal/dal. Litter mates were then used as controls for our studies. Two protocols were carried out. First, wounds were inflicted with a 5 mm punch biopsy. Control wounds were treated with vehicle whereas test wounds were treated with various preparations of a nitric oxide agent (ProliNO) which released proline as well as nitric oxide. We found that the level of ProliNO used was toxic in that it inhibited wound healing. Importantly, there was no observable difference between WT and dal/dal mice. Another protocol we tried is the two-stage skin tumorigenesis model. Again, we found no difference between WT and daql/dal in their production of papillomas. Thus, we had to conclude that under these conditions, prolidaase activity was not a limiting factor.Despite these disappointing results on wound healing, there has been some exciting news from our collaborators in Italy. Dr. Antonella Pavia at the University of Pavia has found some striking skeletal abnormalities in the dal/dal mice. They have decreased femoral length, and decreased cortical and trabecular area of the tibia metaphysic and diaphysis, Clearly, the skeletal development was affected by the deficiency in prolidase. Dr. Forlino is pursuing these studies as a model for the skeletal abnormalities in humans with prolidase deficiency.