Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov 25;116(22):4720-30.
doi: 10.1182/blood-2010-05-286872. Epub 2010 Aug 25.

Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization

Amber N Stratman  1 , Amy E Schwindt, Kristine M Malotte, George E Davis
Affiliations

Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization

Amber N Stratman et al. Blood. .

Abstract

Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)-lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs. Using either soluble receptor traps consisting of the extracellular ligand-binding domains of platelet-derived growth factor receptor β, epidermal growth factor receptor (EGFR), and ErbB4 receptors or blocking antibodies directed to platelet-derived growth factor (PDGF)-BB, or heparin-binding EGF-like growth factor (HB-EGF), we show that both of these EC-derived ligands are required to control pericyte motility, proliferation, and recruitment along the EC tube ablumenal surface. Blockade of pericyte recruitment causes a lack of basement membrane matrix deposition and, concomitantly, increased vessel widths. Combined inhibition of PDGF-BB and HB-EGF-induced signaling in quail embryos leads to reduced pericyte recruitment to EC tubes, decreased basement membrane matrix deposition, increased vessel widths, and vascular hemorrhage phenotypes in vivo, in support of our findings in vitro. In conclusion, we report a dual role for EC-derived PDGF-BB and HB-EGF in controlling pericyte recruitment to EC-lined tubes during developmental vascularization events.

PubMed Disclaimer

Figures

Figure 1
Figure 1
ECs are required to induce pericyte motility and proliferation in 3D collagen matrices during EC-pericyte tube coassembly. Nuclear GFP–labeled pericytes (nuc-pericytes) were incorporated into 3D collagen matrices in the presence or absence of ECs and allowed to undergo morphogenesis over a period of 120 hours. Real-time video analysis was performed to assess pericyte motility and proliferative rates over 0-72 hours or 72-120 hours. (A) Representative images of tracking analysis that were done with nuc-pericytes over 0-72 or 72-120 hours demonstrates the requirement of ECs to induce pericyte motility in 3D matrices. (B-C) From the tracking data generated in A, the average distance pericytes moved from their point of origin and the average pericyte velocity was measured for both 0-72 hours (B) or 72-120 hours (C). Both of these measures display impaired pericyte motility and decreased pericyte velocity over time. Furthermore, in the absence of ECs, the pericyte proliferative rate is reduced over both time periods, as quantified by the total number of pericytes per high-powered field over time (B-C). n ≥ 5; P ≤ .01.
Figure 2
Figure 2
Addition of soluble PDGFRβ, EGFR, and ErbB4 receptor traps leads to decreased pericyte recruitment and proliferation. GFP-pericytes were allowed to coassemble with ECs for a period of 72 hours, at which time cultures were fixed for further analysis. (A) Addition of soluble PDGFRβ, EGFR, and ErbB4 receptor traps (50 μg/mL) as individual molecules leads to approximately 60% of pericytes being associated with EC tubes and 40% being nonassociated. However, under conditions in which these soluble proteins are added in combination, only 30%-40% of pericytes are associated with EC tubes, and 60%-70% are nonassociated. Data are reported as percent pericyte association. (B) After treatment with soluble protein inhibitors, there is a dramatic increase in EC tube width in all conditions with marked disruption of pericyte recruitment. (C) Addition of these soluble protein inhibitors also leads to decreased pericyte proliferation, as quantified by assessing the total number of GFP-pericytes per high-powered field. (D) Representative images of EC-pericyte coassembly are shown in which pericytes are GFP-labeled and ECs are CD31-immunostained red. Associated pericytes are denoted by arrows; nonassociated pericytes are denoted by arrowheads. Bar equals 20 μm. n ≥ 5; P ≤ .01. *Significance from control conditions. +Significance from individual factor addition.
Figure 3
Figure 3
PDGF-BB– and HB-EGF–specific neutralizing antibodies lead to decreased pericyte recruitment to EC tubes and decreased pericyte proliferation. ECs and GFP-pericytes were allowed to coassemble for a period of 72 hours in the presence or absence of neutralizing antibodies to PDGF-BB and HB-EGF. A control neutralizing antibody directed to IL-6 was also used. Each of the antibodies was added at 50 μg/mL. After 72 hours, cultures were fixed for analysis of pericyte recruitment, EC tube width, and pericyte proliferation. (A) Under conditions of either HB-EGF or PDGF-BB neutralization individually, there is a 20%-30% decrease in the number of pericytes associated with EC tubes. When neutralizing antibodies are added to HB-EGF and PDGF-BB in combination, nearly 80% of pericytes are nonassociated with EC tubes. Data are reported as percent pericyte association. (B) EC tube width was measured in conjunction with pericyte association, demonstrating a dramatic width increase of EC tubes in conditions of disrupted pericyte recruitment. (C) Blockade of PDGF-BB and HB-EGF leads to decreased pericyte proliferation, as measured by assessing the total number of GFP-pericytes per high-powered field. n ≥ 5; P ≤ .01. *Significance from control conditions. +Significant from individual factor addition.
Figure 4
Figure 4
Blockade of PDGF-BB and HB-EGF, using neutralizing antibodies or soluble protein fragments, leads to abrogated EC-induced pericyte motility responses in 3D collagen matrices. Nuc-pericytes were allowed to coassemble with ECs either in the presence or absence of neutralizing antibodies to PDGF-BB and HB-EGF or soluble protein fragments to PDGFRβ and the HB-EGF receptor, ErbB4. Real-time video analysis was carried out to determine the effect of these molecules on pericyte motility. (A) The movement of nuc-pericytes was tracked, using MetaMorph software, with measures of pericyte average velocity, average total distance of movement, and average distance from the origin shown. As demonstrated, disruption of PDGF-BB and HB-EGF signaling in combination leads to blockade of each of the measurements made as markers of pericyte motility in the presence of ECs. (B) Representative images of the nuc-pericyte tracking analysis are shown to demonstrate the inability of pericytes to migrate under conditions of combined PDGF-BB and HB-EGF inhibition. n ≥ 5; P ≤ .01.
Figure 5
Figure 5
Blockade of pericyte recruitment to EC tubes leads to decreased basement membrane matrix deposition. EC/GFP-pericyte cocultures were established for 3 days and fixed for immunostaining analysis. Detergent-free immunostaining methods were used to assess the extracellular deposition of the key basement membrane components: collagen IV (Col IV), laminin (LM), and fibronectin (FN). (A) Quantification of immunostaining intensity is shown, demonstrating the decrease in basement membrane protein deposition under conditions of abrogated pericyte recruitment. (B) Intensity mapping of the representative images from panel C is shown to demonstrate the sites of highest basement membrane protein deposition, based on immunostaining patterns. (C) Representative images of the immunostained EC-pericyte cocultures are shown, with each of the basement membrane proteins in red and GFP-pericytes in green, under control versus PDGFRβ-Fc/EGFR-Fc–treated cultures. As shown by these images, there is decreased basement membrane protein deposition in cultures that have inhibited pericyte recruitment. The final column (CD31/Collagen IV) displays representative overlays of collagen IV (red), with the EC marker, CD31 (blue), and the corresponding pericytes (green) to show the relationship between the 3 structures. Bar equals 20 μm; n ≥ 5; P ≤ .01.
Figure 6
Figure 6
PDGFRβ and EGFR inhibition through the use of chemical inhibitors or neutralizing antibodies in vivo leads to a blockade of pericyte recruitment to EC tubes and concomitant cranial and abdominal hemorrhage phenotypes in developing quail embryos. Two chemical inhibitors and 2 neutralizing antibodies were identified based on their ability to interfere with PDGFR signaling (imatinib and α-PDGF-BB) and EGFR signaling (gefitinib and α-HB-EGF) and administered individually or in combination to quail at 72 hours of embryonic development at a doses of 100nM for the chemical inhibitors and 20 μg/mL for the neutralizing antibodies. The quail were then allowed to develop for 144 hours, at which time the eggs were cracked and the embryos assessed for vascular phenotypes. (A-B) Embryos treated with individual reagents developed mild cranial hemorrhages, while those embryos treated with both gefitinib/imatinib or α-PDGF-BB/HB-EGF, to block PDGFR and EGFR signaling simultaneously, led to more severe hemorrhage phenotypes (Table A). (C-D) CAM tissue from control, gefitinib/imatinib double treatment, and α-PDGF-BB/HB-EGF double treatment embryos was isolated and double stained for the quail EC-specific marker QH1 (green) and PDGFRβ (red). (C) Representative images are shown demonstrating pericyte association with microvascular beds. Arrows denote representative nonassociated pericytes. (D) The number of nonassociated pericytes per high-powered field was quantified, showing an increase in the number of nonassociated pericytes with blood vessels in vivo after treatments to inhibit PDGFR and EGFR signaling. n ≥ 5; P ≤ .01.
Figure 7
Figure 7
Blockade of EC-pericyte interactions in vivo leads to decreased basement membrane deposition and increased EC vessel width. (A) Quail CAM tissue from the controls, gefitinib/imatinib-treated quail embryos and α-PDGF-BB/HB-EGF–treated embryos, was isolated and immunostained for the basement membrane component fibronectin. Quantification of immunostaining intensity of extracellular basement membrane protein deposition displays a decrease in deposition under conditions of inhibited pericyte recruitment, most severely in conditions of combined PDGFR and EGFR inhibition. (B) Representative images of the fibronectin stains are shown, with arrows highlighting areas of decreased levels of extracellular basement membrane protein deposition. Overlays of QH1 staining (EC marker, green) versus fibronectin (red) are included for control versus α-PDGF-BB/HB-EGF treatments. (C) Measurements of EC tube width were done (from QH1 stains of EC tubes), demonstrating increased EC vessel width under conditions of inhibited pericyte recruitment to EC tubes. Furthermore, there was a decrease in the number of EC branch points in CAMs treated with these chemical inhibitors or blocking antibodies, further implicating direct vascular phenotypes. (D) Representative images of CAM tissue stained with the quail EC specific marker, QH1, are shown demonstrating the increased vessel width and decreased branch point phenotypes. Arrowheads indicate the “membrane-ruffled appearance” that was particularly observed in the gefitinib/imatinib condition and correlated with strongly reduced fibronectin deposition and increased vessel widths. n ≥ 5; P ≤ .01. *Significance from control conditions. +Significance from individual factor addition.
See this image and copyright information in PMC

References

    1. Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999;36(1):2–27. - PubMed
    1. Betsholtz C, Karlsson L, Lindahl P. Developmental roles of platelet-derived growth factors. Bioessays. 2001;23(6):494–507. - PubMed
    1. Hellstrom M, Gerhardt H, Kalen M, et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol. 2001;153(3):543–553. - PMC - PubMed
    1. Lindblom P, Gerhardt H, Liebner S, et al. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall. Genes Dev. 2003;17(15):1835–1840. - PMC - PubMed
    1. Betsholtz C, Lindblom P, Gerhardt H. Role of pericytes in vascular morphogenesis. Exs. 2005;(94):115–125. - PubMed

Publication types

Substances