Analysis of the mRNA Expression of C-Type Natriuretic Peptide-3 and Natriuretic Peptide Reseptor-2 in Angiogenesis of Chick Chorioallantoic Membrane: A Descriptive Study

Abstract

The chick chorioallantoic membrane (CAM) is a widely used model to follow the developmental processes in angiogenesis studies. This study aims to describe the putative involvement of CNP-3, the chick homolog of human CNP, and its receptor natriuretic peptide receptor (NPR)-B besides known angiogenic factors and their receptors, vascular endothelial growth factor (VEGF)-A/ vascular endothelial growth factor receptor (VEGFR)-2 and fibroblast growth factor (FGF)-2/fibroblast growth factor receptor (FGFR)-2, in angiogenesis of the CAM. CAM samples between developmental days E7 and E20 were collected for light microscopic and transmission electron microscopic analyses. Expression of CNP-3/NPR-B, VEGF-A/VEGR-2, FGF-2/FGFR-2 mRNA in these CAM samples were also studied between E7 and E20. Light microscopy and electron microscopy analyzes showed that, vascular organization was mostly within the chorionic mesenchyme as endothelial tube-like structures on E7-E8 days. On E9 and later, advanced blood vessels were observed within the mesenchyme. VEGF-A and FGF-2 expression were observed in the early stages of CAM development (E7-E9) with a peak at E8. These molecules showed a second peak at around E16. Co- expression of VEGF-A, FGF-2 and CNP-3 were seen at E16-E20. VEGFR-2 expression was observed between E7-E12, whereas expression of FGFR-2 showed its first peak between E7-E9 and its second peak between E16-20. NPR-B expression, on the other hand, was observed between E7-E20 with its highest level at E16. In conclusion, the results revealed that CNP-3 may have a role in vascular organization via its NPR-B receptor in the later stages, i.e., E16-E20, of CAM development.

Keywords

• CNP
• CNP-3
• NPR-B
• angiogenesis
• chorioallantoic membrane (CAM)

Project Number

DA20/14

Tavuk Embriyosu Koryoallantoik Membranında Görülen Anjiyogenez Sürecinde C-Tipi Natriüretik Peptid-3 ve Natriüretik Peptid Reseptör-B mRNA İfadelerinin Analizi: Tanımlayıcı Bir Çalışma

Abstract

Tavuk koryoallantoik membran (CAM) modeli, anjiyogenez çalışmalarında gelişimsel süreçleri takip etmek için yaygın olarak kullanılan bir modeldir. Bu çalışmada, CAM kullanılarak, embriyonik gelişimin 7. ve 20. günleri (E7 ve E20) arasında görülen anjiyogenez süreci ışık ve transmisyon elektron mikroskop analizleri ile takip edilmiştir. Ayrıca, aynı günlerde alınan CAM örnekleri kullanılarak, anjiyogenez meka-nizmalarında görevli [Vasküler Endotelyal Büyüme Faktörü (VEGF)-A ve Fibroblast Büyüme Faktörü (FGF)-2] ve reseptörlerinin [Vasküler Endotelyal Büyüme Faktörü Reseptörü (VEGFR)-2 ve Fibroblast Büyüme Faktörü (FGFR)-2] yanı sıra, insan C-tipi natriüretik peptid (CNP) homoloğu olan tavuk CNP-3 ve reseptörü natriüretik peptid reseptörü (NPR)-B'nin mRNA ifade düzeylerinin zamana bağımlı değişimi analiz edilmiştir. Işık mikroskobu ve elektron mikroskobu analizleri, endotel tüp benzeri vasküler yapıları yoğun olarak E7-E8 günlerinde ve koryonik mezenşim içinde bulunduğunu göstermiştir. E9-E20 günler sürecinde ise mezenşim içinde yer alan vasküler yapıların progresif gelişimi, stabilizasyonu ve kompleks dallanması gözlenmiştir. Bu vasküler gelişim sürecinde, VEGF-A ve FGF-2 mRNA ifadesi, CAM gelişiminin erken aşamalarında E7 ile E9 arasında ve E8'de pik yapacak şekilde gözlenmiştir. Bu moleküller, CAM gelişiminin geç döne-minde, E16 civarında ikinci bir pik göstermiştir. CNP-3 mRNA ifadesi E16 ile E20 arasında VEGF-A ve FGF-2 mRNA ifadeleri ile eş zamanlı olarak tespit edilmiştir. VEGFR-2 mRNA ifadesi E7-E12 arasında gözlenirken, FGFR-2 mRNA ifadesi ilk pikini E7-E9 arasında ve ikinci pikini E16-20 arasında göstermiştir. NPR-B mRNA ifadesi ise en yüksek seviyesi E16’da olmak üzere E7-E20 arasında gözlenmiştir. Sonuç olarak elde edilen veriler, CNP-3'ün özellikle CAM gelişiminin geç döneminde (E16-E20 günleri sürecinde), NPR-B reseptörü aracı-lığıyla, ileri vasküler organizasyonda rol oynayabileceğini ortaya koymuştur.

Keywords

• CNP
• CNP-3
• NPR-B
• anjiyogenez
• koryoallantoik membran (CAM)

Supporting Institution

Başkent Üniversitesi

Project Number

DA20/14

Thanks

Elektron mikroskop örneklerinin hazırlanmasında teknik desteği için Başkent Üniversitesi Tıp Fakültesi Histoloji ve Embriyoloji Anabilim Dalından Uzman Biyolog Ece Lakşe Coşar’a teşekkür ederiz.

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