The fetal porcine aorta and mesenteric acellular matrix as small-caliber tissue engineering vessels and microvasculature scaffold.

BACKGROUND: The extracellular matrix (ECM) is characterized by not only well-preserved scaffolds of organs and vascularized tissues, but also by extremely low immunogenicity during allo- or xeno-implantation. This study aimed to establish a model of a composite microvasculature network scaffold with a small-caliber-dominant vascular pedicle by decellularizing fetal porcine aorta and the conterminous mesentery. METHODS: The aorta and the conterminous mesenteric vascular system originating from the inferior mesenteric artery were harvested from fetal pigs at late gestation. All of the cellular components were removed by sequential treatment with Triton X-100 and sodium dodecyl sulfate. After the degree of decellularization was assessed, the fetal porcine aorta and mesenteric acellular matrix (FPAMAM) were transplanted into dogs. RESULTS: Gross and histologic examination demonstrated the removal of cellular constituents with preservation of ECM architecture, including macrochannels and microchannels. The residual DNA content in the FPAMAM was less than 2 %. The aorta and microchannels were perfused well, and the fetal porcine aorta had good patency for more than 3 months. CONCLUSIONS: The integrity of the FPAMAM provided a scaffold for the reconstruction of a rich vascular network with numerous segmentally radiating branches. Decellularized fetal porcine vascular tissue might be a potential alternative for xenogeneic transplantation based on its optimized properties and low immunogenicity. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The role of Frizzled-4 mutations in familial exudative vitreoretinopathy and Coats disease.

AIM: The aim of this study is to assess the role of Frizzled-4 (FZD4) in familial exudative vitreoretinopathy (FEVR) and Coats disease. METHODS: Tissue samples were collected for DNA extraction and automated DNA sequencing of the two coding exons of FZD4 in both directions. Cases carrying a FZD4 mutation and demonstrating extreme disease severity were selected for direct automated sequencing of all coding exons of LRP5, NDP and TSPAN12. Clinical data were obtained for the purpose of identifying genotype-phenotype correlations. RESULTS: 68 probands were diagnosed as having autosomal dominant or sporadic FEVR. Eleven FZD4 mutations (five missense, three deletions, one insertion, two nonsense) were identified. Six of these mutations are novel, and none were found in 346 control chromosomes. In 16 cases of Coats disease, one polymorphism combination was found in two samples: no mutations were detected. No genotype-phenotype correlation emerged. Three severely affected cases with FZD4 mutations failed to show additional mutations in the three other FEVR genes. CONCLUSION: The authors identified 12 FEVR probands with FZD4 mutations. FZD4 mutation screening can be a useful tool especially in mild or atypical cases of FEVR. Germ-line mutations in FZD4 do not appear to be a common cause of Coats disease.

Severe retinopathy of prematurity associated with FZD4 mutations.

PURPOSE: To determine whether mutations in the FZD4 gene are a risk factor for developing severe ROP. METHODS: Three Canadian tertiary care centers recruited premature infants prospectively and retrospectively, and assigned affectation status based on the maximum degree of severity of ROP recorded in both eyes. Mutation screening of the FZD4 gene was performed using direct sequencing. All sequence changes were evaluated for functional significance. RESULTS: Two novel FZD4 mutations (Ala370Gly or Lys203Asn) were identified in two infants from the severe ROP group (n=71). No mutation was detected in the mild to no ROP group (n=33), and the two novel mutations were absent in 173 random Caucasian samples. Mutation Ala370Gly was also found in one sibling and one parent of the affected infant, but no signs of familial exudative vitreoretinopathy (FEVR), a condition with phenotypic overlap with ROP known to be caused by FZD4 mutations, were present in either family member. CONCLUSIONS: Mutations in the FZD4 gene in this group of premature infants supports a role for the FZD4 pathway in the development of severe ROP and accounts for approximately 3% of severe ROP in Caucasian premature infants.

Phenotypic overlap of familial exudative vitreoretinopathy (FEVR) with persistent fetal vasculature (PFV) caused by FZD4 mutations in two distinct pedigrees.

PURPOSE: To describe a severe familial exudative vitreoretinopathy (FEVR) phenotype seen in infancy that resembles persistent fetal vasculature (PFV) caused by mutations in the FZD4 gene in two pedigrees with high intrafamilial variability. METHODS: Three infants presented with features compatible with bilateral PFV. Eye examinations from the affected children and their relatives were reviewed retrospectively (follow-up:18 months-9 years). Mutation screening was performed using direct sequencing of the FZD4, LRP5 and NDP genes. RESULTS: Bilateral retinal folds extending from the optic nerve to the inferotemporal aspect of the lens mimicing PFV were observed in two of the three affected children before the age of two months. The third child was examined at birth, and the avascular peripheral retina treated with diode laser within one week of age, with subsequent arrest of the disease process. A FZD4 mutation, M493_W494del, was identified in one affected child in pedigree 1, and a novel missense mutation, I114T, was detected in 2 affected children in pedigree 2; while no mutations were found in NDP or LRP5 genes in the 3 affected children. In both pedigrees, at least one affected relative was asymptomatic and failed to show the characteristic avascular changes of FEVR. CONCLUSIONS: The clinical features in the three children and their relatives with a documented FZD4 mutation support the previous reports of a high degree of intrafamilial and interfamilial variability in FEVR. In extreme cases with very early onset, the development of a retinal fold can mimic PFV, a non-hereditary condition with rare exception.

Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.