Extracellular Matrix and Stress Substrates: the Role of Prolidase

我们需要一种氨酰基脯氨酸二肽酶缺乏症的动物模型，它反映了患有这种遗传性代谢紊乱的人类的表型。人类疾病的特征是伤口愈合不良，下肢溃疡，骨骼异常和智力迟钝。然而，这些在人类身上的发现并不一致。在某些家系中，具有相同生化表型的同胞可能具有不同的临床表现。为了进行这些研究，我们与蒙大拿州大福尔斯麦克劳克林医学研究所的特蕾莎古恩博士建立了合作关系。她描述了一种毛色异常的小鼠品系（dal/dal）。她培育了一群纯合子dal/dal小鼠，然后在编码脯氨酰二肽酶的pepd基因中发现了一个4 bp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，我们获得了用C3 H繁殖dal/dal的杂合子，并获得了F2动物，对这些动物进行了红细胞氨酰基脯氨酸二肽酶检测，以确定WT和dal/dal。然后将同窝仔用作我们研究的对照。进行了两个方案。首先，用5 mm穿孔活组织检查造成伤口。对照伤口用载体处理，而测试伤口用释放脯氨酸以及一氧化氮的各种一氧化氮试剂（ProliNO）制剂处理。我们发现所使用的ProliNO的水平是有毒的，因为它抑制伤口愈合。重要的是，WT和dal/dal小鼠之间没有可观察到的差异。我们尝试的另一个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，氨脯氨酸酶活性不是限制因素。尽管伤口愈合的结果令人失望，但我们在意大利的合作者还是带来了一些令人兴奋的消息。帕维亚大学的Antonella Pavia博士在dal/dal小鼠中发现了一些惊人的骨骼异常。他们股骨长度缩短，胫骨干骺端和骨干的皮质和骨小梁面积减少，显然，骨发育受到氨酰基脯氨酸二肽酶缺乏的影响。Forlino博士正在进行这些研究，作为氨酰基脯氨酸二肽酶缺乏症患者骨骼异常的模型。

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.