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© The Rockefeller University Press,
0021-9525/2001//543 $5.00
The Journal of Cell Biology, Volume 153, Number 3,
, 2001 543-554
Original Article |
Lack of Pericytes Leads to Endothelial Hyperplasia and Abnormal Vascular Morphogenesis
christer.betsholtz{at}medkem.gu.se
The association of pericytes (PCs) to newly formed blood vessels has been suggested to regulate endothelial cell (EC) proliferation, survival, migration, differentiation, and vascular branching. Here, we addressed these issues using PDGF-B– and PDGF receptor-β (PDGFR-β)–deficient mice as in vivo models of brain angiogenesis in the absence of PCs. Quantitative morphological analysis showed that these mutants have normal microvessel density, length, and number of branch points. However, absence of PCs correlates with endothelial hyperplasia, increased capillary diameter, abnormal EC shape and ultrastructure, changed cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability. Brain endothelial hyperplasia was observed already at embryonic day (E) 11.5 and persisted throughout development. From E 13.5, vascular endothelial growth factor-A (VEGF-A) and other genes responsive to metabolic stress became upregulated, suggesting that the abnormal microvessel architecture has systemic metabolic consequences. VEGF-A upregulation correlated temporally with the occurrence of vascular abnormalities in the placenta and dilation of the heart. Thus, although PC deficiency appears to have direct effects on EC number before E 13.5, the subsequent increased VEGF-A levels may further abrogate microvessel architecture, promote vascular permeability, and contribute to formation of the edematous phenotype observed in late gestation PDGF-B and PDGFR-β knock out embryos.
Key Words: mice • angiogenesis • pericytes • platelet-derived growth factor B • vascular endothelial growth factor
© 2001 The Rockefeller University Press
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Introduction |
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The PC/vSMCs are probably induced de novo from the mesenchyme surrounding the endothelium (Beck and D'Amore 1997). In vitro experiments implicate TGF-β in this process (Antonelli-Orlidge et al. 1989; Sato and Rifkin 1989; Hirschi et al. 1998). In addition, targeted mutagenesis in mice implicates TGF-β signaling in the process of PC/vSMC development (Li et al. 1999; Yang et al. 1999; Oh et al. 2000). An early role for angiopoietin-1 and its receptor Tie-2 in the formation of PC/vSMCs is suggested from the phenotypes of angiopoietin-1 and Tie-2 knock outs, which both exhibit reduced embryonic PC/vSMC formation (Suri et al. 1996; Patan 1998). In addition, humans with a point mutation in the tie-2 gene activating its tyrosine kinase exhibit venous malformations with regionally reduced vSMC coating (Vikkula et al. 1996).
After induction, the population of PC/vSMCs is expanded and recruited to angiogenic sprouts by means of proliferation and migration (Nicosia and Villaschi 1995; Beck and D'Amore 1997; Benjamin et al. 1998). This longitudinal spreading of PC/vSMCs along growing blood vessels depends on PDGF-B and PDGF receptor-β (PDGFR-β) (Lindahl et al. 1997; Hellstrom et al. 1999). In the developing mouse embryo, PDGF-B is produced by the endothelium, whereas PDGFR-β is expressed by developing PC/vSMCs (Holmgren et al. 1991; Lindahl et al. 1997). Targeted disruption of PDGF-B or PDGFR-β genes both lead to formation of blood vessels lacking PC/vSMCs. Chimeric analysis of wild-type and PDGFR-β–null cells has shown that PDGFR-β is cell autonomously required in the PC/vSMC lineage (Crosby et al. 1998; Lindahl et al. 1998). Together, these data suggest that longitudinal recruitment of PC/vSMCs is controlled by paracrine signaling involving PDGF-B released from endothelial cells (ECs) to act on PDGFR-β expressed by neighboring PC/vSMCs.
The PDGF-B– and PDGFR-β–null mutations are homozygously lethal at birth. The immediate cause of lethality appears to be a sudden onset of microvascular hemorrhaging and edema formation. However, multiple cardiovascular abnormalities are apparent from earlier embryonic age, including cardiac dilation and hypertrabeculation, abnormal kidney glomeruli lacking mesangial cells, an abnormal placenta labyrinthine layer, and dilation of large and small caliber blood vessels (Levéen et al. 1994; Soriano 1994; Lindahl et al. 1997; Hellstrom et al. 1999; Ohlsson et al. 1999). The microvascular hemorrhaging is preceded by the formation of microaneurysms, which are morphologically similar to the characteristic lesions in human diabetic microangiopathy (Cogan et al. 1961). It is possible that all phenotypes observed in PDGF-B and PDGFR-β knock outs are caused by a reduction in the abundance of PC/vSMCs, but both the normal role of PCs in microvessels and the pathogenesis of the cardiovascular changes in PDGF-B and PDGFR-β knock outs remain unclear. It is likely, however, that PCs influence EC behavior. PCs have been suggested to have a role in endothelial sprouting, and the localization of PCs to capillary branch points suggests a role in vascular branching morphogenesis (Nehls et al. 1994). It has been proposed that PCs negatively regulate EC proliferation in vitro (Orlidge and D'Amore 1987; Hirschi et al. 1999). On the other hand, PCs appear to promote EC survival in the pruning process during formation of the retinal vasculature and in tumor vessels after experimental withdrawal of the vascular endothelial growth factor-A (VEGF-A) (Benjamin et al. 1998, Benjamin et al. 1999). Based on these findings, the lack of PCs in vivo would either be predicted to lead to an increase in EC proliferation, resulting in EC hyperplasia, or to a decreased EC survival, resulting in EC hypoplasia. Although mechanistically conflicting, both results could lead to vascular dysfunction and cause the phenotypes observed in PDGF-B– and PDGFR-β–null embryos. The present investigation addresses the role of PCs making use of the unique properties of PDGF-B– and PDGFR-β–deficient mice as an in vivo model of brain angiogenesis in the absence of PCs (Lindahl et al. 1997; Hellstrom et al. 1999). We show that PCs are not determining microvessel density, length, or branching, but are involved in the regulation of EC number and morphology and microvessel architecture.
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Materials and Methods |
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EC Quantification and Capillary Morphometry
Capillary EC nuclei were counted on 5-µm paraffin sections of brains fixed in 4% paraformaldehyde. Sections were stained with propidium iodide and biotinylated isolectin BS-I (L-2140; Sigma-Aldrich) as described (Hellstrom et al. 1999), except for the use of streptavidin Alexa 488 (S-11223; Molecular Probes) at a final concentration of 2 µg/ml. EC nuclei were counted using the 60x objective on a Nikon Eclipse E1000 microscope with a fluorescent filter allowing simultaneous monitoring of red and green signals. For morphometric analysis, 14-µm sections were stained with isolectin BS-I and horseradish peroxidase (Hellstrom et al. 1999) and analyzed using the 10x objective on an Optronics charge-coupled device camera and associated software. Capillary blood vessel length and branching were quantified on 15 photographic areas. The capillary diameter was quantified on 15 photographic areas at 200 sites.
Immunohistochemistry
The following antibodies were used at the dilutions indicated: polyclonal rabbit anticaveolin-1 (1:500; Transduction Laboratories), polyclonal rabbit antiaquaporin-4 (1:100; Chemicon), polyclonal rabbit anticlaudin-1 (1:500; Zymed), polyclonal rabbit antioccludin (1:200, clone Z-T22; Zymed), polyclonal rabbit anti–ZO-1 (1:500; Zymed), rat monoclonal anti–VE-cadherin (culture supernatant; provided by Dr. Dietmar Vestweber, University of Muenster, Muenster, Germany), FITC-conjugated polyclonal sheep antifibronectin (1:100; Biotrend), polyclonal rabbit anticlaudin-5 (1:1,000; production described in Liebner et al. 2000). Embryonic brains were dissected and directly frozen in Tissue-Tek OCT (Miles Scientific). Cryosections were cut at 12 µm, mounted on glass slides, air dried, and fixed in ice-cold ethanol for 5 min followed by 1 min in acetone at room temperature. Sections were rinsed and incubated with the primary and appropriate secondary antibodies as described (Gerhardt et al. 1996). Slides were coverslipped using Moviol and analyzed using a Leica TCS NT confocal laser scanning microscope (Leica). For direct comparison between wild-type and knock out brains, sections were mounted pairwise on the slides. Photomultiplier settings were identical for image acquisition from knock out and wild-type specimen.
Electron Microscopy
Embryos were fixed by perfusion through the heart of 2.5% glutaraldehyde in Hank's modified salt solution buffer. Brains were immersion fixed in the same fixative for an additional 4 h, washed, postfixed, dehydrated, and processed for transmission electron microscopy as described (Gerhardt et al. 1996). For freeze fracture analysis, tissue specimens were fixed as above, cryoprotected for freeze fracture in 30% glycerol, and frozen in nitrogen slush (–210°C). Subsequent freeze fracture of the tissue and analysis by electron microscopy was done as described (Kniesel et al. 1996).
RNA Analyses
Total RNA was prepared from snap frozen brain tissue using the RNeasy midi kit (75144; QIAGEN) according to the manufacturer's protocol. For Northern blot analysis, 10 µg RNA was used for Northern blot analysis by the NorthernMax Kit (1940; Ambion). Mouse probes for β-actin, GRP-78, and glucose transporter 1 (Glut-1) and rat probes for phosphoglycerate kinase (PGK) and lactate dehydrogynase (LDH; probes provided by Dr. Peter Carmeliet, Center for Transgene Technology and Gene Therapy, Leuven, Belgium) were randomly labeled using Megaprime DNA labeling system (RPN 1607; Amersham Pharmacia Biotech) according to the manufacturer's protocols. For RNase Protection analysis, 30 µg of RNA was used in the RPA III kit (1414; Ambion), and polyacrylamide gels were exposed and scanned using a BAS-1500 PhosphoImager (Fuji). Riboprobes were prepared using RNA polymerase (Promega) and [32P]UTP (Amersham Pharmacia Biotech) according to the manufacturer's protocol. Mouse cDNA fragments used as templates to make the probes are as follows: Flt-1, 437 bp, NM_002019.1; Tie1, EcoRI fragment of 
400 bp, X80764; Flk-1, 725 bp, X70842. β-Actin template (Ambion), which generates a 250-bp protected fragment, was used as an internal control for quantification.
VEGF-A Measurements
Tissues were snap frozen and kept at –80°C until analysis and subsequently lysed and homogenized in 150 mM NaCl, 66 mM EDTA, 10 mM Tris-HCL (pH 7.4), 0,4% sodiumdeoxycholate, 1% NP-40, and protease inhibitor cocktail (P-2714; Sigma-Aldrich). The VEGF-A ELISA detection kit (MMV00; R&D Systems) was used to quantify VEGF-A protein from tissue lysates. The plates were read in a Kinetic microplate reader (Molecular Devices). The VEGF-A concentration was normalized against the total amount of protein in the sample using protein assay (500-0006; Bio-Rad Laboratories).
Statistics and Image Processing
The two-tailed paired Student's t test was used for all statistical calculation. Pictures were assembled and processed in Adobe® PhotoshopTM, v5.5.
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Results |
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60% in the number of nuclei per capillary cross section in PDGF-B and PDGFR-β knock out brains from E 11.5 on (Fig. 1 c). This increase persisted throughout prenatal development and correlated with the lack of PCs in the PDGF-B and PDGFR-β knock out brains seen at E 11.5 and throughout prenatal development (Hellstrom et al. 1999; data not shown). Studies of longitudinally sectioned capillaries and serial sections showed that EC nuclear shape was not altered in mutants (not shown). Thus, an increased nuclear count reflects EC hyperplasia. EC hyperplasia was not observed in the perineural vascular plexus surrounding the brain (data not shown) where PCs are still present in the PDGF-B and PDGFR-β knock out embryos (Lindahl et al. 1997; Hellstrom et al. 1999). BrdU and TdT-mediated dUTP-biotin nick end labeling staining was analyzed at several time points between E 10.5 and E 18.5, but no significant differences between wild-type and mutant embryos were revealed (data not shown). This excludes a simultaneous large increase in proliferation and apoptosis, but the sensitivity of the analysis does not allow small changes in cell number that may arise over several cell cycles to be correctly determined.
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We performed RNase Protection assays for the endothelial receptor tyrosine kinases VEGFR-1/Flt-1, VEGFR-2/Flk-1, and Tie1 to assess if these transcripts were altered in the mutants. Total RNA was analyzed at E 17.5 in the wild-type and PDGFR-β knock out brains and normalized against β-actin. In the PDGFR-β knock out brain, the VEGFR-1 signal was 146% compared with wild type (p-value = 0.077), VEGFR-2 signal was 179% compared with wild type (p-value = 0.002), and Tie1 signal was 259% compared with wild type (p-value = 0.02). There is good correlation between the magnitudes of the expression of VEGFR-1 and -2 and the EC hyperplasia, suggesting that the expression of VEGFR-1 and -2 is not altered in the absence of PCs. The larger increase in Tie1 compared with VEGFR-1 and -2 expression in mutants is compatible with a previous report that Tie1-driven LacZ expression is increased in the PDGF-B knock out (Lindahl et al. 1997).
Abnormal Blood Vessel Morphogenesis in PDGF-B and PDGFR-β Knock Outs
The EC hyperplasia prompted us to assess vessel diameter and EC–EC interaction. The mean blood vessel diameter was increased by 25% (P < 0.001) in the PDGFR-β knock out compared with wild type but was highly variable (Fig. 2 a). The blood vessels of the PDGF-B and PDGFR-β knock outs exhibited regional dilations along the blood vessel length, which was not seen in the wild type. (Fig. 2b and Fig. c; data not shown).
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Increased Levels of VEGF-A in PDGF-B and PDGFR-β Knock Out Brains and Livers
We performed ELISA quantification of VEGF-A protein in brain and liver at several time points throughout development to determine its relation to EC hyperplasia in the PDGF-B and PDGFR-β knock out brains. The liver was added to the analysis since liver PCs are recruited independently of PDGF-B and PDGFR-β (Hellstrom et al. 1999). In PDGF-B and PDGFR-β knock outs, VEGF-A levels were progressively increased in both the brain and the liver from E 13.5 until birth (Fig. 3, a and b). By Northern blot analysis, the VEGF-A RNA level was also upregulated at E 16.5 in the PDGF-B knock out (data not shown).
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Redistribution of VE-Cadherin and Occludin in ECs of PDGF-B and PDGFR-β Knock Out Brains
To further analyze the basis for vascular leakage in PDGF-B and PDGFR-β knock outs, we performed a detailed analysis of adherens and tight junction proteins by immunolabeling and confocal laser scanning microscopy. VE-cadherin, the major cadherin involved in EC–EC contact (Breier et al. 1996; Navarro et al. 1998), exhibited reduced junctional staining and increased cytoplasmic distribution in the PDGFR-β knock out compared with wild type at E 18.5 (Fig. 7, a and b), but no difference was seen at E 12.5 (data not shown). Occludin, an integral part of the tight junction (Furuse et al. 1993), showed less distinct junctional staining in PDGFR-β knock out ECs compared with wild type at E 18.5 (Fig. 7d and Fig. e). This difference was not seen at E 12.5 (data not shown). Similar changes in cellular distribution of VE-cadherin and occludin were found in brain vessels of E 18.5 PDGF-B knock outs (data not shown). Analysis of the tight junction proteins ZO-1 and claudin-1 and -5 revealed no differences in junctional localization or staining intensity in PDGF-B or PDGFR-β knock outs at E 18.5 or E 12.5 (Fig. 7g and Fig. h; data not shown). The microaneurysms typical for the PDGF-B and PDGFR-β knock out brains exhibited highly abnormal staining for all the above mentioned proteins (Fig. 7c, Fig. f, and Fig. i), most likely coupled to the extreme endothelial morphology at these sites.
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Discussion |
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Pericytic Influence on Capillary Morphogenesis
Although it is plausible that the EC hyperplasia in PDGF-B and PDGFR-β embryos may contribute to an increased capillary diameter, the reduced EC junctional circumference and the increased EC cytoplasmic thickness suggest that ECs are more crowded in the microvessel wall. An average 25% increase in vessel diameter, as seen in the mutants, would result on average in a two times increase in tension in the vascular wall at constant blood pressure. This could, by itself or in combination with the lack of the structural support from PCs, promote microaneurysm formation. A schematic illustration of the capillary EC phenotype in the PDGF-B/PDGFR-β knock outs is shown in Fig. 10.
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Endothelial Hyperplasia Precedes VEGF-A Upregulation
VEGF-A is a potent endothelial mitogen critical for embryogenesis (for review see Ferrara 1999). A narrow range of VEGF concentration is required for normal cardiovascular development, since a 50% reduction or a two- to threefold increase in VEGF-A expression both lead to embryonic lethality in mice (Carmeliet et al. 1996; Ferrara et al. 1996; Gerber et al. 1999; Miquerol et al. 2000). We found that VEGF-A levels were upregulated more than twofold in PDGF-B and PDGFR-β knock out embryos at late gestation. However, VEGF-A was not significantly upregulated at the earliest time points of EC hyperplasia in mutants but became progressively elevated from E 13.5 on. Although an increased expression of VEGF-A does not seem to explain the EC hyperplasia in the knock outs before E 13.5, VEGF-A may support the hyperplastic state later. In a model of regulated VEGF expression in tumors, Benjamin and co-workers (1999) demonstrated that VEGF withdrawal leads to selective EC apoptosis and degeneration of tumor vessels lacking PC/vSMCs. Their study suggests that either VEGF or contact with PC/vSMCs is sufficient to promote EC survival in tumor vessels. In analogy with their study, the absence of PCs in PDGF-B and PDGFR-β knock out brains may be compensated by increased VEGF-A levels, allowing for survival of the increased number of ECs.
Upregulation of VEGF-A and Other Markers of Metabolic Stress Suggests Systemic Defects in PDGF-B and PDGFR-β Knock Outs
VEGF-A is induced by hypoxia and hypoglycemia both at the RNA and protein levels (Shweiki et al. 1992; Stein et al. 1998). Upregulation of VEGF-A, Glut-1, GRP-78, LDH, and PGK suggests that PDGF-B and PDGFR-β knock out embryos are influenced by metabolic stress. Several causes of hypoxia and/or hypoglycemia in these knock outs can be envisioned. First, it might originate from low tissue oxygenation due to malfunctioning microvessels. Second, the labyrinthine layer of the placenta has a reduced number of PCs and trophoblasts in PDGF-B and PDGFR-β knock outs, which results in dilated embryonic vessels and in a reduced fetal–maternal interface (Ohlsson et al. 1999). This might result in inefficient exchange between the fetal and maternal blood vessels, leading to shortage of oxygen and nutrients and accumulation of waste products in mutant embryos. It is interesting to note that the earliest morphological signs of placenta defects in mutants occur at E 13.5, coincident with the increased VEGF-A levels. Third, the PDGF-B and PDGFR-β knock out hearts are dilated and hypotrophic, which may have systemic consequences (Levéen et al. 1994; Hellstrom et al. 1999). A scenario for the pathogenic cause–effect relationships in PDGF-B and PDGFR-β knock outs is outlined in Fig. 9 b.
Vascular Leakage Is Accompanied by Elevated Vesiculation and Altered Junctional Architecture
The edema in late PDGF-B and PDGFR-β mutant embryos prompted us to analyze markers of EC junctions and transcellular transport. Vesicular transport by caveolae is a major pathway for transcellular permeability (for review see Feng et al. 1999). The increase in caveolae and caveolin-1 staining seen in PDGF-B and PDGFR-β mutants suggests that this may cause (a portion of) the vascular leakage in these embryos.
Paracellular transport is controlled by tight and adherens junctions. Tight junctions are built up by lipids and transmembrane proteins, which are anchored to the cytoskeleton (for review see Kniesel and Wolburg 2000). Freeze fracture analysis of the endothelial tight junctions revealed no differences between the wild type and the PDGF-B knock out. However, the transmembrane protein, occludin, was redistributed and showed less distinct tight junctional staining in the PDGF-B and PDGFR-β knock outs. The series of events leading to the developmental acquisition of blood–brain barrier properties in ECs has been described to involve the shift of occludin from cytoplasmic pools to the tight junctions (Gerhardt et al. 1999). Accordingly, the elevated cytoplasmic and reduced junctional localization of occludin in the knock outs may be interpreted as a sign of less mature tight junctions. Similarly, the adherens junction protein VE-cadherin was partially redistributed from a junctional to a cytoplasmic location. Together, this suggests that tight and adherens junctions are formed in knock outs but do not exhibit the full degree of differentiation. It is known that VEGF-A promotes both paracellular and transcellular permeability in ECs by increasing the number of caveolae and by rearranging occludin and VE-cadherin (Vasile et al. 1999). It is therefore possible that the increased levels of VEGF-A directly induce vascular leakage in the PDGF-B and PDGFR-β knock outs. This idea is strengthened by the fact that there are no differences in caveolin-1, occludin, or VE-cadherin staining at E 12.5, when VEGF-A is not yet upregulated, and no increase in vascular leakage has been observed in the mutants. Furthermore, high expression of VEGF-A has been correlated with leaky blood vessels in several experimental and pathological conditions (for reviews see Dvorak et al. 1999; Ferrara and Alitalo 1999).
The vascular leakage in the PDGF-B and PDGFR-β knock out brains is likely a major cause of edema and glial swelling. This could be the trigger of the upregulation of the astrocyte-specific water channel aquaporin-4 (Wen et al. 1999) in glial cells surrounding the abnormal blood vessels in PDGF-B and PDGFR-β knock outs, which is consistent with aquaporin-4 upregulation in other conditions where vascular leakage and brain edema is prominent (Vajda et al. 2000).
In summary, our analysis suggests that PCs regulate EC number during embryonic angiogenesis. Elevated VEGF-A levels, possibly secondary to systemic metabolic stress in PDGF-B and PDGFR-β knock outs, may also contribute to the vascular abnormalities seen in these embryos, in particular to the increased vascular leakage observed at late gestation. Our analyses suggest that the pathogenesis of vascular abnormalities also in other mouse mutants is complex and may depend on a combination of local cues, e.g., mechanical properties or signaling events, and secondary systemic influences (Fig. 9 b). The number of mouse knock outs with lethal cardiovascular syndromes available for analysis is steadily increasing, but for most of these, the understanding is still rather poor. The determination of pathogenic chains of events in these mutants is challenging but may also have important implications for the understanding of microvessel disease in humans, such as diabetic microangiopathy.
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Acknowledgments |
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This work was supported by the Swedish Cancer Foundation, The Novo Nordisk Foundation, IngaBritt and Arne Lundberg Foundation, and Göran Gustafsson Foundation (to C. Betsholtz); the Deutsche Forschungsgemeinschaft (grant Wo 215/14-1; to H. Wolburg); the Graduiertenkolleg Neurobiologie of the University of Tuebingen and the European Molecular Biology Organization (to H. Gerhardt); and the Ludwig Institute for Cancer Research (to M. Hellstrom and C. Betsholtz).
Submitted: 21 September 2000
Revised: 26 January 2001
Accepted: 13 March 2001
Abbreviations used in this paper: E, embryonic day; EC, endothelial cell; Glut-1, glucose transporter 1; LDH, lactate dehydrogynase; PC, pericyte; PDGFR-β, PDGF receptor-β; PGK, phosphoglycerate kinase; VEGF-A, vascular endothelial growth factor-A; vSMC, vascular smooth muscle cell.
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