Ccn2 mutant mice exhibit vascular defectsCcn2 mutant mice exhibit perinatal lethality due to a severechondrodysplasia [27]. CCN2 expression in developing bloodvessels raised the possibility of an additional role in vasculardevelopment. Ccn22/2 embryos were examined to investigatethis possibility. No overt differences between Ccn2 mutants andWT littermates were apparent during the initial formation of thevasculature from E9.5–E13.5 （data not shown）。 Moreover,placentas were normal in appearance, weight, and vascularitythroughout development （Figure S1B,C, and data not shown）。
However, beginning at E14.5, minor enlargement of vessels wasobserved in mutants （Figure S1D,E）， which became morepronounced at later stages （Figure 2A,B）。 Local edema was seenin E18.5 mutant dermis （Figure 2C,D）。 Immunofluorescenceanalysis of the vSMC marker smooth muscle actin （SMA） andPECAM （CD-31） did not reveal obvious evidence that SMCcoverage of large vessels was affected in mutants （Figure S1F–I）。
However, comparison of hematoxylin and eosin-stained sections ofthe aorta at thoracic and lumbar levels from E16.5 embryosshowed defects in the organization of the tunica media （Fig. 2E–H）。 In WT embryos, SMCs had a spindle-like morphology andwere circumferentially oriented around the vessel lumen in distinctlayers （Figure 2E,G）。 In mutants, SMCs failed to adopt thisspindle-like morphology, were more heterogeneous in size, andwere not organized into distinct layers （Figure 2F,H）。 The largevessel phenotype will be reported in more detail elsewhere. Herewe focus on the microvascular phenotype.
Morphological examination （Figure S1J,K） revealed thatarterial-venous identity appeared to be maintained in mutants（see also Figure S1H,I）。 Ephrin B2 （expressed on arterial elements）and EphB4 （preferentially expressed on veins） staining demonstratedno defects in arterial-venous identity （Figure S1L,M, anddata not shown）。 However, inspection of E18.5 dermal microvasculaturerevealed evidence of defective remodeling in Ccn2mutants. Consistent with a defect in remodeling, vessel densitywas increased in Ccn2 mutants （Figure 2I–L and Figure S2A–C）。
Moreover, mutant capillaries had multiple protrusions along theirsurfaces （Figure 2M,N）。 Electron microscopy revealed numerousluminal and abluminal protrusions in mutant capillaries, consistentwith the confocal analysis （Figure 2O,P）。
CCN2 mutants exhibit defects in vascular remodelingPCNA labeling and TUNEL analyses were performed to assesswhether defects in proliferation and/or survival might contributeto the microvascular abnormalities in Ccn2 mutants. No differenceswere detected in mutants in comparison to WT littermates （FigureS2D–G）。 During vascular remodeling, immature vascular bedsbecome less dense, arterioles become smaller in diameter thanvenules, and pericytes form stable associations with endothelialtubes [34]. Angiopoetin 1 （Ang1） is required for stabilizingendothelial-pericyte interactions and is expressed primarily bymural cells [35]. Ang1 mRNA levels were diminished in Ccn22/2skin （Figure S2H）。 No differences were detected in levels of expression of Tie2, the endothelial-specific receptor for Ang1 （datanot shown）。 However, levels of the mRNA encoding the bio-activeVEGF isoform 164 were elevated in mutants （Figure S2I）。
Versican is the principal chondroitin sulfate proteoglycan in bloodvessels and exists in at least four isoforms, V0, V1, V2, and V3[36]. Embryonic endothelial cells express more V0 than otherisoforms, and V0 expression declines during vascular maturation[37]. No differences were seen in levels of versican V1 in Ccn2mutants and WT littermates （Figure S2J）； however, Ccn2 mutantsexhibited increased levels of V0 （Figure S2K）。 Therefore, the lossof Ccn2 leads to diminished expression of vessel maturation markerAng1 and elevated expression of markers of immature vasculature,indicative of a potential defect in vascular remodeling.
The vascular phenotype in Ccn2 mutants bears some resemblanceto mice lacking platelet-derived growth factor-B （PDGF-B）or its receptor, PDGFRb [38,39]. In particular, defective pericyterecruitment is seen in these mice. Therefore, we examined pericyterecruitment in Ccn2 mutants. Pericytes, which express NG2 anddesmin, become associated with small diameter vessels duringvessel maturation [40]. Consistent with the gene expressionanalysis described above, confocal analysis of desmin expressionrevealed incomplete coverage of microvessels by pericytes in thedermis of Ccn2 mutants at E16.5 and E18.5 （Figure 3A–C; datanot shown）。 Similar results were seen for NG2 expression in thelung liver, and brain microvasculature （Figure 3D–F, and data notshown）。 Thus, the loss of CCN2 affects the microvasculature inmultiple tissues. Flow cytometric analysis of lung, liver, and brainsamples from E16.5 embryos for cells negative for the endothelialcell marker PECAM, but expressing the pericyte markers NG2and PDGFRb [41] revealed normal numbers of endothelial cellsand pericytes in Ccn2 mutants （Figure S3, and data not shown）。
This suggests that the reduced pericyte coverage in Ccn2 mutants isnot caused by a decrease in pericyte number or migration, butpossibly by defects in the ability of pericytes to make stableassociations and elongate along endothelial cells in Ccn2 mutantmice.
Confocal analysis of E16.5 dermal and lung microvasculatureco-stained with NG2, desmin, and PECAM supports thispossibility. NG2 staining demonstrated that pericytes associatedwith WT vessels were in close contact with the capillaryendothelium and appeared elongated along the endothelial surface（Figure 3G）。 In contrast, pericytes associated with capillaries inmutants were more rounded and exhibited less elongation（Figure 3H）。 Immunostaining with desmin also suggested a defectin pericyte association with endothelial cells in mutants. In WTcapillaries, pericytes were elongated and covered the surface ofendothelial tubes （Figure 3I,J and Figure S4A,B）。 In contrast,pericytes on mutant capillaries were rarely elongated, and vesselcoverage was incomplete （Figure 3K,L and Figure S4C,D）。 Takentogether, these findings indicate that the ability of pericytes to formstable associations with microvascular endothelium is defective inCcn2 mutants.
