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, 307 (1), C25-38

Type-2 Pericytes Participate in Normal and Tumoral Angiogenesis

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Type-2 Pericytes Participate in Normal and Tumoral Angiogenesis

Alexander Birbrair et al. Am J Physiol Cell Physiol.

Abstract

Tissue growth and function depend on vascularization, and vascular insufficiency or excess exacerbates many human diseases. Identification of the biological processes involved in angiogenesis will dictate strategies to modulate reduced or excessive vessel formation. We examine the essential role of pericytes. Their heterogeneous morphology, distribution, origins, and physiology have been described. Using double-transgenic Nestin-GFP/NG2-DsRed mice, we identified two pericyte subsets. We found that Nestin-GFP(-)/NG2-DsRed(+) (type-1) and Nestin-GFP(+)/NG2-DsRed(+) (type-2) pericytes attach to the walls of small and large blood vessels in vivo; in vitro, type-2, but not type-1, pericytes spark endothelial cells to form new vessels. Matrigel assay showed that only type-2 pericytes participate in normal angiogenesis. Moreover, when cancer cells were transplanted into Nestin-GFP/NG2-DsRed mice, type-1 pericytes did not penetrate the tumor, while type-2 pericytes were recruited during its angiogenesis. As inhibition of angiogenesis is a promising strategy in cancer therapy, type-2 pericytes may provide a cellular target susceptible to signaling and pharmacological manipulation in treating malignancy. This work also reports the potential of type-2 pericytes to improve blood perfusion in ischemic hindlimbs, indicating their potential for treating ischemic illnesses.

Keywords: angiogenesis; pericytes; stem cells; tumor.

Figures

Fig. 1.
Fig. 1.
Two pericyte subtypes are found in capillaries and larger blood vessels. A and B: pericyte subtypes in mesenteric vessels and skeletal muscle from Nestin-GFP/NG2-DsRed mice. A: pericytes surround mesenteric blood vessels. All panels show the same area for different channels (Nestin-GFP, NG2-DsRed, merged fluorescence, bright-field, and merged fluorescence and bright-field images). B: pericytes surround vessels in skeletal muscle. Muscle longitudinal sections show small blood vessels with CD31+ endothelial cells surrounded by NG2-DsRed+ pericytes. Red and green arrows indicate Nestin-GFP/NG2-DsRed+ (type-1) and Nestin-GFP+/NG2-DsRed+ (type-2) pericytes, respectively. Blood vessels are labeled by the endothelial cell marker CD31. All panels show the same area for different channels (Nestin-GFP, NG2-DsRed, Hoechst, CD31 staining, bright-field, merged fluorescence, and merged fluorescence and bright-field images). C: pericyte markers platelet-derived growth factor receptor-β (PDGFRβ) and CD146 colocalize with skeletal muscle interstitial Nestin-GFP-/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells. Panels show identical muscle areas from top to bottom: PDGFRβ or CD146 (orange), NG2-DsRed (red), Nestin-GFP+ (green), Hoechst (blue), bright-field, and merged images. Red arrow indicates type-1 pericytes (Nestin-GFP/NG2-DsRed+); green arrow indicates type-2 pericytes (Nestin-GFP+/NG2-DsRed+). Scale bar, 20 μm.
Fig. 2.
Fig. 2.
Only type-2 pericytes are angiogenic in vitro. A: procedures for isolation of mononucleated cells from skeletal muscle of Nestin-GFP/β-actin-DsRed double-transgenic (Tg) mice. B: representative flow cytometry dot plot showing green fluorescent protein (GFP) vs. allophycocyanin (APC) fluorescence, with the gate set using cells isolated from wild-type mice. C and D: representative dot plots showing GFP vs. APC fluorescence using mononucleated cells from skeletal muscle of Nestin-GFP/β-actin-DsRed mice, unlabeled and after labeling with NG2 proteoglycan APC antibody. Two cell populations were isolated by sorting: Nestin-GFP/NG2-APC+/β-actin-DsRed+ (type-1 pericytes, yellow) and Nestin-GFP+/NG2-APC+/β-actin-DsRed+ (type-2 pericytes, green). All cells are DsRed+ and can be tracked in vitro after they are mixed with human umbilical vein endothelial cells (HUVECs). E: in vitro Matrigel assay containing a mixture of HUVECs with type-1 or type-2 DsRed+ pericytes sorted from skeletal muscle of Nestin-GFP/β-actin-DsRed mice. DsRed fluorescence, bright-field, and merged images are shown. After 10 days, only HUVECs cultured with type-2 pericytes formed stable vessel-like networks. F: percentage of HUVECs forming vessel-like structures in coculture with type-1 or type-2 pericytes. Values are means ± SE.
Fig. 3.
Fig. 3.
Matrigel plug assay shows that only type-2 pericytes are angiogenic in vivo. A: transplantation scheme of an in vivo Matrigel plug assay. A mixture of HUVECs and either type-1 or type-2 pericytes sorted from skeletal muscle are embedded in Matrigel and implanted subcutaneously into nude mice. Matrigel plug is recovered after 2 wk for analysis. B: gross anatomy of freshly removed Matrigel plug with type-1 or type-2 pericytes 2 wk after implantation. Type-2 pericytes, together with endothelial cells, form blood vessels in vivo. C: β-actin-DsRed fluorescence around blood vessels in a Matrigel plug tracks type-2 pericytes in vivo.
Fig. 4.
Fig. 4.
Nestin-GFP+/NG2-DsRed+, but not Nestin-GFP/NG2-DsRed+, cells, invade brain tumor mass. A: intracranial injection of allograft tumor cells and brain preparation for histology. Growing G26-H2 murine glioblastoma cells were injected into the brain of a Nestin-GFP/NG2-DsRed double-transgenic mouse. B: representative image of a Nestin-GFP/NG2-DsRed mouse brain coronal section showing margin of a G26-H2 murine brain tumor. Dashed line indicates separation between tumor and normal surrounding tissue. Nestin-GFP+/NG2-DsRed+ cells are shown invading the tumor; Nestin-GFP-/NG2-DsRed+ cells are present only in adjacent healthy tissue. Left: identical areas in the brain section: NG2-DsRed (red), Nestin-GFP+ (green), Hoechst (blue), bright-field, NG2-DsRed (red) and Nestin-GFP+ (green) merged, and all images merged. Right: NG2-DsRed (red), Nestin-GFP+ (green), and Hoechst (blue) merged. Regions in red, yellow, and white boxes show Nestin-GFP/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ in normal tissue and Nestin-GFP+/NG2-DsRed+ cells invading the tumor tissue, respectively; these areas are magnified in C. White arrows indicate Nestin-GFP+/NG2-DsRed+ cells in the tumor. C: magnification of NG2-DsRed+ cells in normal and tumor cells in B. Identical brain tissue areas are shown for different channels: NG2-DsRed (red); Nestin-GFP+ (green); Hoechst (blue); bright-field (BF); merged NG2-DsRed (red) and Nestin-GFP+ (green) (N/N); all images merged; and merged NG2-DsRed (red), Nestin-GFP+ (green), and Hoechst (blue) (N/N/H). In normal tissue, NG2-DsRed+ cells express Nestin-GFP transgene or not, while only NG2-DsRed+/Nestin-GFP+ cells are present in the tumor. D: percentage of Nestin-GFP/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells in normal brain or tumor 35 days after cell implantation (n = 3 preparations). Predominantly type-2 pericytes invade the tumor.
Fig. 5.
Fig. 5.
Type-2 pericytes are recruited during tumor angiogenesis. A: protocol for subcutaneous allograft tumor growth. B16 melanoma cells are injected into Nestin-GFP/NG2-DsRed double-transgenic mice, and tumor plus normal adjacent healthy tissue is surgically removed 2 wk later. B: representative immunofluorescence image of a Nestin-GFP/NG2-DsRed mouse tumor section stained with anti-CD31 antibody illustrating margin of the melanoma tumor and surrounding normal tissue. Vertical dashed line indicates separation between tumor and normal skeletal muscle. The same tissue area is shown for different channels: NG2-DsRed (red), Nestin-GFP+ (green), CD31 (orange or blue), Hoechst (blue), and merged images. Regions selected in red, yellow, and white boxes show type-1 pericytes in normal tissue, type-2 pericytes in normal tissue, and type-2 pericytes associated with tumor blood vessels, respectively. These areas are magnified in C. Only type-2 pericytes are associated with CD31+ blood vessel endothelial cells in the tumor, while type-1 and type-2 pericytes associate with these cells in the normal skeletal muscle. Type-1 pericytes are present only in adjacent healthy tissue. C: magnification of pericytes in normal and tumor tissues in B. Identical tissue areas are shown for different channels: NG2-DsRed (red), Nestin-GFP+ (green), CD31 (orange), Hoechst (blue), bright-field, and all fluorescent merged images. Type-1 and type-2 pericytes are associated with vessels (CD31+) in the normal tissue, while only type-2 pericytes associate with tumor vessels. D: percentage of Nestin-GFP/NG2-DsRed+ (type-1) and Nestin-GFP+/NG2-DsRed+ (type-2) pericytes in normal and cancer tissues 15 days after tumor implantation (n = 3 preparations). Only type-2 pericytes are recruited during tumor blood vessel formation.
Fig. 6.
Fig. 6.
Type-2 pericytes recover blood flow in a mouse model of hindlimb ischemia. A: hindlimb ischemia protocol in nude mice. Femoral artery is ligated and transected, type-1 or type-2 pericytes are intramuscularly injected into the ischemic leg, and limb perfusion is assessed with in vivo MRI angiography. B: high-resolution in vivo MRI. Angiography from mouse hindlimbs right after ligation of the femoral artery (time 0) and after 10 days of treatment. Red circles indicate femoral artery location. C: MRI angiography of hindlimbs below the knee 10 days after treatment. D: new, partially developed femoral artery in ischemic legs of mice injected with type-2 pericytes. Red rectangles indicate location of femoral arteries. E: DsRed and GFP fluorescence around vessels in whole muscles 10 days after type-2 pericyte transplantation. Scale bar, 100 μm. O, occlusion of femoral artery; P1, type-1 pericytes; P2, type-2 pericytes.
Fig. 7.
Fig. 7.
Schematic representation of type-2 pericyte involvement in angiogenesis. A: type-1 (yellow) and type-2 (green) pericytes are associated with blood vessels. We propose that only type-2 pericytes are angiogenic. B: type-2 pericytes participate in angiogenesis associated with ischemic conditions and tumor progression.

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