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. 2011 Feb;178(2):911-23.
doi: 10.1016/j.ajpath.2010.10.012.

Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis

Shuei-Liong Lin  1 , Fan-Chi Chang, Claudia Schrimpf, Yi-Ting Chen, Ching-Fang Wu, Vin-Cent Wu, Wen-Chih Chiang, Frank Kuhnert, Calvin J Kuo, Yung-Ming Chen, Kwan-Dun Wu, Tun-Jun Tsai, Jeremy S Duffield
Affiliations

Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis

Shuei-Liong Lin et al. Am J Pathol. 2011 Feb.

Abstract

Microvascular pericytes and perivascular fibroblasts have recently been identified as the source of scar-producing myofibroblasts that appear after injury of the kidney. We show that cross talk between pericytes and endothelial cells concomitantly dictates development of fibrosis and loss of microvasculature after injury. When either platelet-derived growth factor receptor (R)-β signaling in pericytes or vascular endothelial growth factor (VEGF)R2 signaling in endothelial cells was blocked by circulating soluble receptor ectodomains, both fibrosis and capillary rarefaction were markedly attenuated during progressive kidney injury. Blockade of either receptor-mediated signaling pathway prevented pericyte differentiation and proliferation, but VEGFR2 blockade also attenuated recruitment of inflammatory macrophages throughout disease progression. Whereas injury down-regulated angiogenic VEGF164, the dys-angiogenic isomers VEGF120 and VEGF188 were up-regulated, suggesting that pericyte-myofibroblast differentiation triggers endothelial loss by a switch in secretion of VEGF isomers. These findings link fibrogenesis inextricably with microvascular rarefaction for the first time, add new significance to fibrogenesis, and identify novel therapeutic targets.

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Figures

Figure 1
Figure 1
Selective inhibition of VEGFR2 or platelet-derived growth factor (PDGFR)β signaling by soluble circulating receptors attenuates kidney fibrosis after ureteral obstruction. A, B: Western blots of kidney myofibroblast phospho-PDGFRβ (A) or human umbilical vein endothelial cells phospho-VEGFR2 (B) stimulated by PDGF-BB or VEGF and inhibited by purified sPDGFRβ and sVEGFR2-Fc in vitro. C: Time course of circulating adenovirus-mediated sPDGFRβ and sVEGFR2-Fc production by Western blot analysis of 1 μl of plasma after i.v. administration of adenoviruses (1 × 109 plaque-forming units) to adult Coll-GFP mice 3 days before unilateral ureteral obstruction (UUO) surgery. D–F: Immunofluorescence images (D, E) of day 1 post-UUO Coll-GFP kidneys and Western blot (F) of day 4 post-UUO kidneys from mice with circulating adenovirus-mediated sPDGFRβ, sVEGFR2-Fc, or sFc, detecting phospho-PDGFRβ or phospho-VEGFR2 (arrowheads in D and E). G, H: Low-power fluorescence images of Coll-GFP in kidney sections in control (CON) or day 4 after UUO (g denotes glomerular podocytes) (G), indicating collagen 1 (α1) transcription and interstitial pericytes/perivascular fibroblasts (H), from mice-producing systemic sPDGFRβ, sVEGFR2-Fc, or sFc. I: Western blot of GFP protein or GAPDH levels (upper) or Q-PCR for collagen 1 (α1) transcripts (lower) in control [C] and day 4 after UUO [U] kidney cortex (n = 6/group). J, K: Images of picrosirius red-stained kidney sections for interstitial fibrillar collagens (red) (J), and morphometric quantification of fibrillar collagen from whole sagittal kidney sections (K). L: Q-PCR of transcripts of profibrotic effectors, in kidney cortex day 4 after UUO in mice-producing systemic sPDGFRβ, sVEGFR2-Fc, or sFc. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 6/group).
Figure 2
Figure 2
The platelet-derived growth factor (PDGFR)β or VEGFR2 blockade prevents pericyte proliferation, migration, and differentiation in kidneys after unilateral ureteral obstruction (UUO) injury. A–C: Split-image immunofluorescence micrographs of Coll-GFP+ pericytes/myofibroblasts in control or day 4 after UUO kidneys co-labeled with the pan-cell-cycle marker Ki-67 (A), and quantification of Coll-GFP+, Ki-67+ interstitial cells (arrowheads in A and B), or Ki-67+ tubule epithelial cells (C). D: Immunofluorescence micrographs of endothelial (CD31, red) cell, and Coll-GFP+ cell interactions in mouse kidneys on days 1 and 2 after UUO or control kidneys showing Coll-GFP+ pericyte detachment and migration from capillaries (arrowheads) by day 1 and pericyte population expansion by day 2, only in mice producing circulating sFc protein. E: Graph of Coll-GFP+ interstitial cell number at early timepoints after UUO. F–H: Immunofluorescence micrographs (F) of kidney interstitial αSMA expression (red) by Coll-GFP+ cells, 4 days after UUO or control (a denotes vascular smooth muscle cells of arterioles (arrowhead) Coll-GFP+ cell without αSMA staining). Quantification of αSMA+ interstitial cells (G) and % of αSMA+, Coll-GFP+ interstitial cells (H). *P < 0.05, ***P < 0.001 versus sFc-treated UUO kidney (n = 6/group).
Figure 3
Figure 3
Platelet-derived growth factor (PDGFR)β or VEGFR2 blockade attenuates recruitment of inflammatory macrophages. A–C: Immunofluorescence micrographs showing interstitial F4/80+ macrophages in Coll-GFP control kidneys, or after unilateral ureteral obstruction (UUO) in mice with circulating sFc, sPDGFRβ, or sVEGFR2-Fc. Graphs (B, C) quantifying F4/80+ kidney interstitial macrophages. D–F: Western blot of VCAM1 and ICAM1 and GAPDH expression in whole kidney in control (C) or day 4 after UUO (U). E: Q-PCR of GAPDH-normalized whole kidney transcripts VCAM1, ICAM1, and TNFα from control and day 4 after UUO mice. F: Q-PCR of monocyte chemokine and chemokine receptor transcripts CCL2 (MCP1) and CX3CL1 (fractalkine) and receptors CCR2, CX3CR1. *P < 0.05, and ***P < 0.001 versus sFc-treated UUO kidney (n = 6/group).
Figure 4
Figure 4
VEGFR2 blockade or PDGFRβ blockade prevent microvascular rarefaction but by different mechanisms. A, B: Split panel immunfluorescence micrographs (A) and quantification (B) of endothelial cells of peritubular capillaries expressing the pan-cell-cycle marker Ki-67 in kidneys after unilateral ureteral obstruction (UUO). C: Morphometric quantification of the kidney microvasculature showing angiogenic response to injury, followed by rarefaction of the microvasculature. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal kidney on day (d) 0. D–F: Immunofluorescence micrographs showing kidney microvasculature (red) in Coll-GFP control kidneys or after UUO in mice with circulating sFc, sPDGFRβ, or sVEGFR2-Fc. E and F: Graphs showing morphometric quantification of kidney microvasculature. Note on day 4 sVEGFR2-Fc, but not sPDGRFβ, prevents angiogenesis. G: Graph quantifying peritubular endothelial (CD31+) cell proliferation in control kidneys or day 4 after UUO. **P < 0.01, ***P < 0.001 (n = 6/group).
Figure 5
Figure 5
Kidney injury-mediated switch from VEGF164 to VEGF120 and VEGF188 isoforms is prevented by PDGFRβ blockade and VEGFR2 blockade. A: Q-PCR time course for whole kidney VEGFA transcript expression and the VEGFA splice variants VEGF164, VEGF120, and VEGF188, in days after the unilateral ureteral obstruction (UUO) surgery. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal kidney at day (d) 0. B: Graphs of normalized Q-PCR for VEGF isomer transcripts showing day 10 after UUO, both PDGFRβ blockade (black bars) and VEGFR2 blockade (hatched bars) prevented the injury-induced increase in VEGF120 and VEGF188 transcripts compared to control Fc (white bars). **P < 0.01, and ***P < 0.001 versus sFc-treated UUO kidney; *P < 0.5, and ***P < 0.001 versus CON. C: Time course Western blot for VEGFA isoforms in kidneys after UUO. D: Immunofluorescence images of control and day 4 after UUO kidneys showing VEGFR2 (red) is not expressed by Coll-GFP+ cells. E: Immunofluorescence images of control and day 4 after UUO kidneys showing PDGFRβ is expressed exclusively in Coll-GFP+ pericytes/fibroblasts. F: Q-PCR time course of whole kidney for normalized PDGF-B transcripts in kidneys after UUO. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal kidney at day 0 (n = 5/group).
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