Studies On The Ultrastructure,Somatic Mutation And Molecular Mechanism Of Hypertrophic And Nodular Port Wine Stain

ObjectivePort wine stain(PWS)is a congenital,progressive vascular malformation of human skin involving the superficial vascular plexus that occurs in estimated 3-5 children per 1,000 live births.Since most malformations occur on the face,personality development is adversely influenced in virtually all young patients.PWS is a clinically significant problem in the majority of patients.Many patients with PWS develop hypertrophy and discrete nodularity during their adult life,but the cause remains incompletely understood.In this project,we attempted to probe the molecular mechanism underlying development and progression of this late stage of PWS through the following aspects:(1)characterization of the main ultrastructures of various cell types in hypertrophic and nodular PWS,(2)investigation of activation status of PI3 K signal pathwayto explore their potential roles in the formation of hypertrophic and nodular PWS lesions,and(3)identification of the possible novel genetic alterations that may be underlying the pathological progressions of PWS.This study is toreveal the mechanisms of hypertrophic and nodular PWSand provide essential information for development of new and more effective therapeutic strategies for the treatment of these laser-resistant PWS types.Methods1.A total of 11 hypertrophic,11 nodular PWS and 7 normal control facial surgical leftover specimens were obtained from 16 PWS patients and 7 normal control subjects who were de-identified for this study.Tissue samples were minced to <0.5mm3,fixed in Karnovsky's solution(1% glutaraldehyde and 4% paraformaldehyde in 0.1M phosphate buffer)for 24 hours,and followed by post-fixation in 1% osmium tetroxide.After fixation,all of the samples were dehydrated in series graded ethanol solutions and embedded in an Epon-epoxy mixture.Ultrathin 50 nm thick sections were prepared by using Leica Ultracut 7 for TEM according to standard procedures.The sections were then examined by a TEM(Tecnai G2 Spirit,FEI,Hillsboro,Oregon)operated at 80 kV.An AMT camera system was used for electron microscopy and image capture.2.A total of 21 PWS biopsy samples,including 8 hypertrophic,8 nodular PWS specimens and 5 specimens from adjacent normal skin 0.5 to 1cm away from PWS lesions,were obtained from 13 subjects and de-identified for this study.Two normal adult skin samples were retrieved from the skin biopsy tissue bank from our department to serve as normal controls.Skin specimens were fixed in Bouin's solution overnight.Then they were dehydrated,cleaned,and embedded in paraffin and sectioned to 5 mm thick.The sections were stained with hematoxylin and eosin(H&E)and photographed under an optical microscope.Routine immunohistochemistry and Western Blotprocedures were used to investigate activation status of PKC?,PI3 K,PDPK1 and PLC-? and protein levels of PP2 A and DAG to explore their potential roles in the formation of hypertrophic and nodular PWS lesions.3.A total of surgical-removed,de-identified 4 hypertrophic,4 nodular PWS specimens and 4 normal control skins adjacent to the lesions were obtained from 4 PWS subjects for this study.The genomic DNA was isolated and purified from the fresh or frozen specimens using DNA Mini Kit(OMEGA),followed by construction of genomic DNA sequencing libraries.The complete coding sequence of the whole genome was captured and enriched using a solution-phase Agilent SureSelect Human All Exon V5(50M)(Agilent Technologies,Inc).The magnitude of enrichment and size of insertion were estimated using Agilent 2100 Bioanalyzer(Agilent DNA 1000 Reagents)and quantitative PCR.The exome libraries were loaded on the Illumina Hiseq 4000 instrument for sequencing.Mutation calls were performed using followed by the SAMtools.Results1.We found in that PWS endothelial cells,pericytes and fibroblasts exhibited a hyperactive status which was recognized by the presence of large numbers of rough endoplasmic reticulum,free ribosomes,Golgi complexes,enlarged mitochondria and vesicles in the cytoplasm.Hypertrophied collagenous bundles were particularly noted in the papillary dermis and surrounding PWS blood vessels.Furthermore,the enlargement of tonofibrils and clustering of melanosomes were more frequently observed in the basal layer of keratinocytes.The epidermal-dermal junctions were degenerative as evidenced by a thin and disrupted basal lamina and less density in hemidesmosomes in nodular PWS as compared to normal skin.2.We found phosphorylated levels of PKC?,PI3 K,PDPK1 and PLC-? and protein levels of PP2 A and DAG showed moderate increases in the endothelial cells of hypertrophic PWS as compared to the adjacent normal skin.These increases extended throughout the entire stroma of blood vessels in PWS nodules.Many proliferating cells,such as fibroblasts,also showed strong activation of PKC?,PI3 K,PDPK1 and PLC-? and up regulations of PP2 A and DAG in nodular PWS lesions.Our data showed there is aberrant activation of PKC?,PI3 K,PDPK1 and PLC-? and up regulation of PP2 A and DAG in endothelial cells only in hypertrophic PWS areas,but presenting in entire vasculatures and surrounding fibroblasts in PWS nodules.3.We identified various missense mutations in many genes through WES,including Trim69(c.825C>T/p.T161M),Muc6(p.Thr1807Ser/c.5420C>G)and Muc3a(c.1951A>T/ p.Ser651Cys;c.1952G>C/p.Ser651Thr;c.1973C>T/p.Ser658Leu;c.2014T>C/ p.Phe672Leu),in PWS hypertrophic and nodular samples but not in controls.In addition,the missense mutation in Igfn1(c.5907A>G/p.I2008M)and two other synonymous mutations inMapk7(T>C,Chr17:19285524)and Znf528(T>C,Chr19: 42729848)genes were identified only in PWS nodules but not hypertrophic and control samples.Conclusion1.We concluded that the entire physiological milieu of skin underwent progressively pathological development in PWS.Our data suggested that hyperactive endothelial cells,pericytes and fibroblasts directly accounted for proliferation of PWS blood vessels and their stroma,resulting in soft tissue hypertrophy and nodularity development.2.Our data suggest that both the PKC? and PI3 K signaling pathways contribute to the development of hypertrophy and nodularity in adult PWS.3.Our data implicate that missense mutations of Trim69(c.825C>T/p.T161M),Muc6(p.Thr1807Ser/c.5420C>G)and Muc3a(c.1951A>T/p.Ser651Cys;c.1952G>C/ p.Ser651Thr;c.1973C>T/p.Ser658Leu;c.2014T>C/p.Phe672Leu)may contribute the hypertrophic development of PWS.In addition,the missense mutation in Igfn1(c.5907A>G/p.I2008M)may play a role in PWS nodular formation.