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Review
, 56 (5), 392-402

Molecular Control of Capillary Morphogenesis and Maturation by Recognition and Remodeling of the Extracellular Matrix: Functional Roles of Endothelial Cells and Pericytes in Health and Disease

Affiliations
Review

Molecular Control of Capillary Morphogenesis and Maturation by Recognition and Remodeling of the Extracellular Matrix: Functional Roles of Endothelial Cells and Pericytes in Health and Disease

George E Davis et al. Connect Tissue Res.

Abstract

This review addresses fundamental mechanisms underlying how capillaries form in three-dimensional extracellular matrices and how endothelial cells (ECs) and pericytes co-assemble to form capillary networks. In addition to playing a critical role in supplying oxygen and nutrients to tissues, recent work suggests that blood vessels supply important signals to facilitate tissue development. Here, we hypothesize that another major function of capillaries is to supply signals to suppress major disease mechanisms including inflammation, infection, thrombosis, hemorrhage, edema, ischemic injury, fibrosis, autoimmune disease and tumor growth/progression. Capillary dysfunction plays a key pathogenic role in many human diseases, and thus, this suppressing function may be attenuated and central toward the initiation and progression of disease. We describe how capillaries form through creation of EC-lined tube networks and vascular guidance tunnels in 3D extracellular matrices. Pericytes recruit to the abluminal EC tube surface within these tunnel spaces, and work together to assemble the vascular basement membrane matrix. These processes occur under serum-free conditions in 3D collagen or fibrin matrices and in response to five key growth factors which are stem cell factor, interleukin-3, stromal-derived factor-1α, fibroblast growth factor-2 and insulin. In addition, we identified a key role for EC-derived platelet-derived growth factor-BB and heparin-binding epidermal growth factor in pericyte recruitment and proliferation to promote EC-pericyte tube co-assembly and vascular basement membrane matrix deposition. A molecular understanding of capillary morphogenesis and maturation should lead to novel therapeutic strategies to repair capillary dysfunction in major human disease contexts including cancer and diabetes.

Keywords: Basement membrane assembly; capillary morphogenesis; endothelial cells; extracellular matrix; pericytes.

Figures

Figure 1
Figure 1. Hypothesis: Capillary tube networks directly suppress major pathogenic mechanisms of disease
We propose the hypothesis that capillary tube networks (primarily composed of EC tubes and associated pericytes) provide signals to adjacent parenchymal and supporting cells within tissue stromal spaces to suppress major mechanisms of disease including cancer initiation and progression. These basic disease mechanisms are also direct contributors to cancer development (arrows). Many human diseases show evidence of capillary dysfunction and we propose that the disease processes directly result as a consequence of this microvascular dysfunction. Capillary dysfunction is a major pathogenic feature of key diseases including diabetes, cancer, obesity and Alzheimer's disease.
Figure 2
Figure 2. Time course of multicellular EC tube assembly in 3D collagen matrices
(A) ECs were seeded as single cells within 3D collagen matrices (mimicking the developmental process of vasculogenesis) and form tubes in response to the “Factors”. They were fixed, stained, and photographed at the indicated time points. Marked EC tube morphogenesis is observed over time. Bar equals 100 μm. (B) GFP- and mCherry-labeled ECs were mixed together at a 1:1 ratio to reveal multicellular EC tube assembly after 72 hr of culture. Cultures were photographed under fluorescence, images were overlaid and the lower panel also shows nuclear staining after the addition of Hoechst dye. Bar equals 100 μm.
Figure 3
Figure 3. Molecular events controlling EC lumen formation during “Factor”-induced EC tube assembly in 3D collagen matrices
ECs were seeded as single cells and allowed to form tubes over 120 hr prior to fixation and processing for transmission electron microscopy (A) or plastic thin sectioning (B) to demonstrate EC lumen formation following cross-sectioning of 3D collagen gels. Black arrows indicate the EC apical surface; white arrows indicate EC junctional contacts, Col I indicates the 3D collagen type I matrix. Key regulatory steps that control the EC lumen formation process are highlighted in the right panel. Bar equals 10 μm (A); Bar equals 100 μm (B).
Figure 4
Figure 4. Capillary tube assembly in 3D collagen matrices and role of vascular guidance tunnels in EC-pericyte tube co-assembly
(A) ECs were co-cultured with GFP-pericytes and at the indicated times, cultures were fixed with paraformaldehyde and stained using anti-collagen type I (Col I) antibodies, and were photographed using light or fluorescent microscopy. EC tubes form, vascular guidance tunnels appear and pericytes are observed to recruit to these tubes on the EC tube abluminal surface within vascular guidance tunnels. Arrowheads indicate the borders of vascular guidance tunnels. Bar equals 50 μm. (B) ECs were co-cultured with GFP-pericytes and at the indicated times, cultures were fixed, immunostained with anti-collagen type I antibodies and photographed using fluorescence microscopy. Marked recruitment of pericytes is observed to EC-lined tubes which are present within vascular guidance tunnels over time. Arrowheads indicate the borders of vascular guidance tunnels. Bar equals 100 μm.
Figure 5
Figure 5. Pericyte motility is observed along EC tubes within vascular guidance tunnels in 3D matrices
(A) mRFP-labeled ECs and GFP-pericytes were co-cultured and videos were made of EC tubulogenesis, pericyte recruitment and pericyte motility along EC tubes over time. Still images from these videos are shown at the indicated times of culture and reveal marked pericyte motility along the abluminal EC tube surface. Note that EC tube remodeling is also occurring while pericytes are moving along the tubes. Bar equals 50 μm. (B) EC and GFP-pericytes were co-cultured and after 120 hr, cultures were fixed and stained with anti-CD31 and anti-collagen type I (Col I) antibodies. Note that pericytes are observed on the EC tube abluminal surface and this recruitment is observed within vascular guidance tunnels (arrowheads indicate the borders of these tunnels), which are generated as a result of the EC tubulogenic process. Bar equals 50 μm.
Figure 6
Figure 6. EC-pericyte tube co-assembly leads to vascular basement membrane matrix deposition
(A) ECs alone or ECs and GFP-pericytes were cultured for 120 hr and then fixed and stained for CD31 or collagen type IV (Col IV), a key basement membrane component. Fixed cultures were not permeabilized with detergent, so that only collagen type IV that was deposited extracellularly is observed. Note that marked basement membrane deposition, as indicated by collagen type IV deposition, is observed only when ECs and pericytes are cultured together. Bar equals 50 μm. (B) EC-pericyte co-cultures were examined by transmission electron microscopy and vascular basement membrane deposition is observed between the two cell types (black arrows). P indicates pericytes, EC indicates endothelial cells, and L indicates the luminal space. Left image- Bar equals 0.5 μm; Right image- Bar equals 1 μm.
Figure 7
Figure 7. Molecular control of human capillary tube morphogenesis and maturation in 3D matrices
Schematic diagram showing key molecular regulators of EC tubulogenesis and pericyte recruitment to EC tubes which control capillary network formation and maturation. Dynamic and polarized EC-pericyte interactions within vascular guidance tunnels results in abluminal vascular basement membrane matrix assembly. EC tubulogenesis is driven by growth factor-dependent signals secondary to a special combination of five growth factors (“Factors”) which are SCF, IL-3, SDF-1α, FGF-2 and Insulin. VEGF can prime ECs in an upstream step to facilitate their responsiveness to the “Factors”. These “Factors” stimulate an integrin-, MT1-MMP-, and Rho GTPase-dependent signaling cascade which controls the development of EC tube networks and vascular guidance tunnels in 3D matrices. These networks produce PDGF-BB and HB-EGF to induce recruitment and proliferation of pericytes and together ECs and pericytes co-assemble within tunnel spaces to co-contribute and deposit the vascular basement membrane along the abluminal EC tube surface in between the two cell types.

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