Fibrin and Collagen Differentially Regulate Human Dermal Microvascular Endothelial Cell Integrins: Stabilization of αv/β3 mRNA by Fibrin1  Xiaodong Feng, Richard A.F. Clark, Dennis Galanakis, Marcia G. Tonnesen  Journal of Investigative Dermatology  Volume 113, Issue 6, Pages 913-919 (December 1999) DOI: 10.1046/j.1523-1747.1999.00786.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Integrin β3 subunit is strongly expressed on blood vessels in day 5 but not in day 7 porcine cutaneous wounds. Cryosections of porcine wounds were immunoprobed with monoclonal antibodies to the integrin subunits αv (a, b) and β3 (c, d) in day 5 wounds (a, c) and in day 7 wounds (b, d). Scale bar: 10 μm. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of integrin subunit mRNAs by HDMEC cultured on immobilized type I collagen, fibronectin, and gelatin. Total RNA was extracted from HDMEC cultured on immobilized type I collagen, fibronectin, and gelatin for 4 h, 8 h, and 24 h. Total RNA was sequentially probed with human integrin cDNA for αv, β3, α2, and β1. Uniformity of gel loading was monitored by ultraviolet light examination of the gel stained with ethidium bromide (data not shown). Uniformity of gel loading and uniformity of RNA transfer to the membrane were demonstrated by hybridization of the same blot with a 32P-labeled probe for 28s ribosomal RNA. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Fibrin and collagen three-dimensional gels differentially regulate mRNA levels of integrin subunits αv and β3. HDMEC cultured on immobilized collagen were overlaid by fibrin or collagen gels for 4 h, 8 h, and 24 h. Total RNA was probed sequentially by human integrin αv and β3 cDNA. (a) Northern blots. (b) Northern blots at 24 h, analyzed by densitometric scanning. Open bars represent collagen and striped bars represent fibrin gel overlay cultures. Uniformity of gel loading was monitored by ultraviolet light examination of the gel stained with ethidium bromide (data not shown). Uniformity of gel loading and uniformity of RNA transfer to the membrane were demonstrated by hybridization of the same blot with a 32P-labeled probe for 28s ribosomal RNA. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 αv and β3 mRNA levels after stimulation by bFGF or VEGF are greater in HDMEC overlaid by fibrin compared with collagen. HDMEC cultured on immobilized collagen were overlaid by fibrin gel or collagen gel, with or without bFGF (50 ng per ml) or VEGF (100 ng per ml) for 24 h. Total RNA was probed sequentially with human integrin αv and β3 cDNA. (a) Northern blots. (b) Northern blots analyzed by densitometric scanning. Open and striped bars represent cultures overlaid by collagen gel and fibrin gel, respectively. Uniformity of gel loading was monitored by ultraviolet light examination of the gel stained with ethidium bromide (data not shown). Uniformity of gel loading and uniformity of RNA transfer to the membrane were demonstrated by hybridization of the same blot with a 32P-labeled probe for 28s ribosomal RNA. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Integrin β3 subunit mRNA is unstable compared with mRNA of subunits αv, α2, and β1. After 24 h of culture on immobilized collagen in EBM with 1% normal human serum and 1% bovine serum albumin, HDMEC were treated with 60 μM 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole (DRB), a RNA transcription initiation inhibitor. Total RNA was isolated at 0 h, 4 h, 12 h, and 24 h and probed with αv, β3, α2, and β1 cDNA. (a) Northern blots. (b) Densitometric scans of northern blots. Diamond, circle with cross, triangle, and square represent mRNA levels of αv, β3, α2, and β1, respectively. Uniformity of gel loading was monitored by ultraviolet light examination of the gel stained with ethidium bromide (data not shown). Uniformity of gel loading and uniformity of RNA transfer to the membrane were demonstrated by hybridization of the same blot with a 32P-labeled probe for 28s ribosomal RNA. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Fibrin enhances the stability of HDMEC integrin subunit αv and β3 mRNA compared with collagen. After 24 h of culture on immobilized collagen in EBM with 1% normal human serum and 1% bovine serum albumin, HDMEC were overlaid by fibrin or collagen gels and treated with 60 μM 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole (DRB), a RNA transcription initiation inhibitor. Total RNA was isolated at 0 h, 12 h, and 24 h and probed with αv and β3 cDNA. (a) Northern blots. (b) Densitometric scans of northern blots. Diamond and square represent αv and β3 mRNA levels of HDMEC overlaid by fibrin and collagen, respectively. Uniformity of gel loading was monitored by ultraviolet light examination of the gel stained with ethidium bromide (data not shown). Uniformity of gel loading and uniformity of RNA transfer to the membrane were demonstrated by hybridization of the same blot with a 32P-labeled probe for 28s ribosomal RNA. Journal of Investigative Dermatology 1999 113, 913-919DOI: (10.1046/j.1523-1747.1999.00786.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions