Further characterization of ATP6V0A2-related autosomal recessive cutis laxa.

Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-ß signalling and increased TGF-ß1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.

Platelet-derived growth factor-B normalizes micromorphology and vessel function in vascular endothelial growth factor-A-induced squamous cell carcinomas.

Vascular endothelial growth factor (VEGF), which is a key regulator of angiogenesis, often induces formation of immature vessels with increased permeability and reduced vessel functionality. Here, we demonstrate that de novo expression of murine (m)VEGF-164 induces malignant and invasive tumor growth of HaCaT keratinocytes. However, the mVEGF-164-induced tumors are ulcerated with a disorganized epithelium that is interrupted by lacunae with limited basement membrane and endothelial cell coverage. Vessel maturation is strongly impaired. Tumor and vessel micromorphology are markedly improved by the combined expression of human platelet-derived growth factor (hPDGF)-B and mVEGF-164. Although tumor size and malignancy are comparable with either mVEGF-164 alone or combined human PDGF-B and mVEGF-164 expression, combined hPDGF-B and mVEGF-164 expression leads to a more solid and compact tumor tissue with a mature functional tumor vasculature and a higher microvessel density, as demonstrated histologically and by dynamic contrast-enhanced magnetic resonance imaging. Treatment of the hPDGF-B- and mVEGF-164-expressing tumors with imatinib mesylate to block PDGF-B signaling reverses this effect. In addition, tumor cell invasion of mVEGF-164 transfectants and mVEGF-164 plus hPDGF-B transfectants in vivo is associated with a marked induction of tumor-derived matrix metalloproteinase-1 and stromal matrix metalloproteinase-9 and -13, as was confirmed in three-dimensional organotypic co-cultures with fibroblasts in vitro. These data clearly demonstrate the need for a concerted action of different growth factors in the establishment of solid tumors with functional vasculature and emphasize the need for a multifactorial therapy.