Roles of PDGFs in the Microvasculature Revealed by Genetic Studies

Lessons from PDGF-B and PDGFRP Knockouts

PDGF-B and PDGFRP null mice both die at late gestation from microcirculatory deficiency involving generalized microvascular hemorrhage and edema [4, 5]. Closer analysis revealed that these mice fail to recruit pericytes to their microvasculature in many but not all organs [6, 7]. The acute cause of death of the PDGF-B and PDGFRP null mice is likely circulatory failure. Live born mutants die from respiratory distress within minutes to hours after birth.

CNS Pericytes

The central nervous system (CNS) microvasculature has a remarkably high density of pericytes in comparison with most other capillary beds. The CNS is also one of the organs most strongly affected by genetic PDGF-B or PDGFRP deficiency. CNS vessels develop by angiogenic sprouting from the perineural vascular plexus. In this process, the invading endothelial cells express PDGF-B and the peri-cytes PDGFRP. In the absence of either ligand or receptor, pericyte co-recruitment largely fails, resulting in <5% of the normal number of brain pericytes. In spite of this cellular deficiency, the endothelial sprouting process proceeds relatively normally, establishing an intracerebral circulation that allows prenatal CNS growth and differentiation. At early stages (E10-14), only mild abnormalities in vessel patterns are observed in associated with a 2-fold increase in endothelial cell number [8]. However, at late gestation, the microvasculature displays more advanced morphological aberrations. Focal dilations and microaneurysms rapidly expand and rupture, leading to multiple sites of hemorrhage and edema. At the cellular level, this correlates with endothelial hyperplasia and abnormal differentiation. Notably, the endothelial luminal membrane surface is oversized and multiple cytoplasmic folds protrude into the vascular lumen. There are also abnormalities in the endothelial adherence junctions and multiple signs of increased vascular permeability. These observations suggest that PDGF-B signaling through PDGFRp is critical for pericyte recruitment to the CNS microvasculature. Moreover, they show that pericytes, in turn, deliver important signals within the microvessel wall that regulate endothelial proliferation and differentiation. At the level of integrated functions, pericytes thereby support microvascular integrity and homeostasis. At a gross anatomical level, pericytes appear to exert control of the capillary diameter.

Mesangial Cell Recruitment

Mesangial cells are pericyte-like cells of the kidney glomerulus. PDGF-B and PDGFRp null embryos both fail to recruit pericytes into the developing glomerulus [4, 5, 9]. As a result, the single capillary loop that invades the nascent glomerular epithelial cleft fails to develop into a complex tuft of vessels. Instead, it dilates into an aneurysm-like structure filling out the glomerular space. During glomerulus development, PDGF-B is expressed in the invading endothelium, whereas the mesangial cells express PDGFRp. A paracrine PDGF-B signal from the endothelium thus promotes mesangial cell co-recruitment into the glomerulus, and the mesangial cells in turn deliver signals that promote glomerular capillary branching and splitting (intussusception). The role of the mesangial cells thus differ somewhat from the brain pericytes, since the overall vessel branching pattern in the brain does not seem to be influenced by the pericytes. However, there are also similarities in that the pericyte/mesangial cell absence in both organs leads to loss of capillary diameter control.

Placenta Pericytes

Placenta pericytes, like glomerular mesangial cells, appear to function in intussusceptive vessel splitting, leading to the formation of complex high-density vascular tufts. Possibly, such specialized pericytes are of particular importance where high vascular densities are needed to fulfill systemic functions (filtration, excretion, absorption etc). Both the kidney and the placenta exert such functions, and the vessels densities are therefore tailored to serve demands of the whole organism, rather than local needs. PDGF-B and PDGFRp null mice show about 50% reduction in the number of placenta pericytes, and an associated impairment of the formation of fetal vessel tufts in the labyrinthine layer of the placenta (where fetal and maternal vessels meet) [10].

Other Locations

Other organs that show a marked (>50%) pericyte deficiency in PDGF-B or PDGFRp null embryos include skin, heart and lung. Interestingly, the major population of liver pericytes, the perisinusoidal cells (Ito cells), is unaffected by PDGF-B or PDGFRp deficiency, and therefore has to develop through other mechanisms. Ito cells also do not express appreciable levels of PDGFRp (mRNA), at least prenatally, and the sinuisoidal endothelium does not detectably express PDGF-B. Also the axial arteries constitute a site at which the number of vSMC/pericytes appears to be unaffected by PDGF-B or PDGFRp deficiency. The origin of these vSMC is believed to be the immature mesenchyme surrounding these vessels. Upon an inductive cue, presumably presented by the endothelial cells, the mes-enchymal cells condensate around the vessel and turn on a SMC differentiation program. This circumferential recruitment of vsMC/pericytes is thus independent of PDGF-B/Rb, whereas the subsequent longitudinal recruitment to new vessels formed by angiogenic sprouting requires paracrine endothelial-to-pericyte signaling using PDGF-B and PDGFRp. Although dispensable for the vSMC induction around the axial arteries, PDGFRp is required for the postnatal maintenance and/or expansion of this vSMC population, as shown by chimeric analysis [11].

Lessons from Conditional PDGF-B Knockouts

Using Cre-lox techniques, vascular endothelium- and neuronal-specific PDGF-B knockouts have been generated [12, 13]. The latter did not reveal observable defects in the brain microvasculature in spite of neurons being a major source of PDGF-B in the CNS. However, the endothelium-specific knockout resulted in pericyte deficiency, confirming the endothelium as the critical source of PDGF-B in pericyte recruitment [12, 14].

Evidence for the Local Importance of Pericytes

The endothelial PDGF-B gene deletion was not complete. Instead, it resulted in a chimeric situation where wildtype vessel segments alternated with PDGF-B deficient segments. In contrast to the PDGF-B null mutants, the endothelium-specific knockouts survived into adulthood, allowing analysis of postnatal angiogenesis. The chimeric nature of the mutant made it possible to spatially correlate pericyte deficiency with microvascular abnormality. This was important, as the role of systemic influences (e.g. heart and placenta defects) on microvessels formation could not be ruled out in the studies of the null mutants. It could therefore be concluded that pericytes control the function of neighboring endothelial cells. Possibly this control involves paracrine growth factors, cell adhesion and junction molecules, and pericyte-derived extracellular matrix molecules.

Retinal Angiogenesis

The survival of the endothelium-specific knockout made it possible to analyze retinal angiogenesis, an early postnatal process. As in other parts of the CNS, pericyte recruitment in the retina was shown to be dependent on endothelial PDGF-B. Studies using neutralizing PDGFRb antibodies confirmed this conclusion [15].

Pericytes and Diabetic Retinopathy

The chimeric nature of the endothelial PDGF-B deletion was inter-individually variable, allowing for analyses of the retinal vascular abnormalities at different states of pericyte density ranging from almost normal (~70% of normal) to near complete deficiency (<10% of normal). Animals where the average pericyte density in the CNS was lower than 50% invariably developed proliferative lesions in the retinas that were reminiscent of diabetic retinopathy. This result was striking since diabetic retinopathy in both humans and animals has been associated with pericyte loss within the microvessel wall. it is not known, however, whether the per-icyte loss constitutes a causal event in the pathogenesis of the retinopathy, or whether it is just correlative with the process. The results from the endothelium-specific PDGF-B knockouts show that independently of a diabetic challenge, pericyte deficiency may cause progressive vascular changes that mimic diabetic retinopathy. The data may also provide an explanation as to why diabetic rodents only develop mild signs of retinopathy and never proliferative disease; pericyte loss in these models is usually substantially lower that 50%.

Lessons from PDGF-B Retention Motif Knockouts

PDGF-B carries a basic stretch of amino acids in its C-terminus referred to as the "retention motif". A similar motif is found in PDGF-A and in different members of the VEGF family, where it is included or excluded by alternative splicing. In PDGF-B the retention motif may remain or be deleted from the precursor depending on proteolytic processing. The basic motifs of the PDGF-B/VEGF family have affinity for heparin and heparan sulphate proteoglycans (HSPGs) and probably serve the same function as analogous motifs in other growth factor families, namely to help generating gradients or depots of factor in the extracellular space. This may ensure graded signaling, or sequestering of the factor in the ECM for release or redistribution by proteolytic cleavage in situations of inflammation.

PDGF-B Retention is Required for Proper Integration of Pericytes in the Vessel Wall

Deletion of the retention motif in PDGF-B resulted in viable mice (PDGF-B ret/ret mice), however, with severe retinopathy [16]. Other signs of microvascular dysfunction were evident as well, including brain microaneurysms and breakage of the blood brain barrier, as well as glomerular defects and proteinuria. All these problems appear to relate to abnormal pericyte and mesangial cell recruitment. Although there was a significant reduction in the number of pericytes in these mice, the degree of reduction did not seem to explain the severity of the phenotype; endothelium-

specific knockouts with a similar decrease in pericyte numbers had much milder retinal and glomerular pathology. The severity of the PDGF-B ret/ret mutants was instead proposed to result from abnormal pericyte integration within the microvessel wall. Notably, pericytes are normally very tightly associated with the abluminal endothelial cell surface. In PDGF-B ret/ret mutants, pericytes were instead partially or fully detached from the endothelial cells. Likely this abrogates the normal communication between the pericytes and the endothelium, leading to a state that resembles the absence of pericytes. in the eye, the dissociated pericytes also form sheets of fibroblast-like cells at the retinal surface, which may contribute to contraction and detachment of the retina.

PDGF-B and the Recruitment of Tumor Pericytes

There are several possible explanations for the detachment of pericytes from the microvessels in PDGF-B ret/ret mice. One possibility is that a graded presentation of PDGF-B is required from the endothelium in order to guide pericyte migration along the abluminal endothelial surface. If the pericyte fails to recognize that the source of PDGF-B is endothelial, they may migrate more randomly, leading eventually to their detachment from the endothelial surface. Another possibility is that a directional presentation of PDGF (i.e. from the endothelium) is needed to polarize the pericyte, forcing unequal distribution of cell and matrix adhesions molecules, or deposition of matrix itself. Such polarization may promote adherence of the pericyte to the endothelium and/or ensure that the pericyte is properly embedded within the microvascular basement membrane. These possibilities, which are not mutually exclusive, are attractive also considering the uneven expression of PDGF-B in the vascular endothelium. The recent finding that the expression is concentrated to specialized endothelial cells situated at the sprout tip (endothelial tip-cells) [17] suggests that the location of the PDGF-B source is critical.

Studies of pericyte recruitment to tumor vessels have provided additional strong support for the idea that directional presentation of PDGF-B is important in the process of pericyte recruitment. In tumors transplanted on PDGF-B ret/ret mice, few pericytes were recruited, and they were partially or fully detached from the endothelium [18]. However, when PDGF levels were compensated by secretion from the tumor cells rather than the endothelium, the number of pericytes in the tumor vessels increased significantly, but the integration of the pericytes in the vascular wall failed. Since the tumors produced the wildtype form of PDGF-B (including the retention motif) it could also be excluded that the integration defect depended on a putative change in the signaling properties of the mutant PDGF-B. Instead, the most plausible explanation is that the PDGF-B protein has to be presented directionally from the endo-thelium, and that the retention motif helps associating the secreted PDGF-B protein to the endothelial surface or within the periendothelial matrix to facilitate such presentation.

Lessons from PDGFRb Signaling Mutants

Using homologous recombination in ES-cells, Soriano and collaborators have generated an extensive allelic series of endogenous signaling mutations in PDGFRb. Mice carrying these mutations show a different range of cardiovascular defects. Taken together, these analyses confirm the importance of PDGFRb signaling in vSMC and pericyte recruitment and in the development of a functional cardiovascular and renal excretory system. However, the analyses also came up with several surprises concerning the relative importance of the different signaling pathways downstream of PDGFRb.

PDGFRb Signaling and Cell Function in Vitro

PDGFRb has been the subject of intense biochemical analysis. It is extensively autophosphorylated upon ligand binding, leading to the formation of up to thirteen phospho-tyrosine residues that engage in coupling to at least ten different SH2-domain containing proteins. These molecules include Src family kinases, PI3 kinase (PI3K), Shc, RasGAP, STATs, Grb2, Grb7, SHP-2, PLCg, and Nck. Their engagement leads to the initiation of several different cellular functions in cultured mesenchymal cells, including proliferation, migration, and extracellular matrix synthesis. in vitro, a certain degree of specificity has been noticed between the signaling pathway engaged and the cellular response. For example, PI3K signaling has been strongly linked to cytoskeletal rearrangements and cell migration. Since analysis of the PDGF-B and PDGFRb null mutants suggests a role for these proteins in pericyte migration and proliferation, it was hoped that the distinct signaling mutations in the endogenous PDGFRb gene would separate between the putative multiple roles of PDGFRb signaling in pericyte/vSMC recruitment. However, as discussed in more detail below, it appears that the various signaling pathways engaged downstream of PDGFRb exert additive rather than distinctive effects in microvascular development.

PDGFRb Unable to Signal Through PI3K and PLCg

Surprisingly, mice homozygous for a mutation that encodes a PDGFRb protein lacking the two PI3K-binding phosphotyrosine residues displayed no overt phenotype [19]. When challenged, however, they display a reduction in the capacity to resolve an experimentally induced tissue edema. Thus, PDGFRb signaling may be implicated in the control of tissue interstitial fluid pressure (IFP). Increased iFP is a general feature of solid tumors and PDGF-B or PDGFRb inhibiting compounds seem to be able to reduce tumor IFP, which in turn facilitates the delivery of substances, including cytostatic drugs, to the tumor [20]. The mechanism by which PDGFRb regulates IFP is not clear, however it is plausible to assume that it involves pericyte or fibroblasts mediated contraction of the ECM. Hence, PDGFRb-mediated PI3K signaling may have its major role in cell contraction in the adult tissues, rather than in cell migration in embryonic development. Recent analysis using embryonic CNS pericyte density as readout also demonstrates that PDGFRb-mediated PI3K signaling takes part in determining the rate of expansion of the pericyte population [21, 22]. Hence PI3K signaling may also be involved in the control of pericyte progenitor proliferation in vivo.

Mice carrying mutant PDGFRb unable to signal through both PI3K and PLCg are likewise without an overtly abnormal phenotype [23]. However, when challenged with an experimental injury to the glomerulus, they display exaggerated pathology. Although it is unclear how the change in pathogenesis relates to defective PDGFRb signaling, it likely involves recruitment or function of mesangial cells, since these are the only cells in the developing glomerulus known to express PDGFRb. Since the overall growth rate of the pericyte population is decreased in the combined PI3K/ PLCg mutants, one might speculate that the proliferation of the mesangial cells is likewise negatively affected, and that this may slow down repair of the injured glomerulus and, in turn, enhance a pathological response.

PDGFRb With Multiple Pathways Defects

Additional mutations removing 5 and 7 phosphotyrosine residues, respectively, in PDGFRb led to overt vascular defects in several organs, in particular when combined with a PDGFRb null allele to reduce the level of PDGFRb expression [21, 22]. These phenotypic abnormalities coincided with a substantial (>50%) reduction in pericyte numbers. The PDGFRb allelic series generated by Soriano and co-workers hence provides a series of mutant animals with a progressive decrease in the pericyte density. This decrease is even further aggravated in animals where the mutant alle-les occur in combination with the PDGFRb null allele.

A mutant has also been created in which the intracellular domain of PDGFRb was replaced with that of PDGFRa [24]. While homozygous carriers of this mutation lack overt phenotype, mice carrying one hybrid receptor and one null allele, showed spontaneous pathology similar to that observed in the mutant lacking 7 phosphotyrosines. This demonstrates that PDGFRa signaling can only partially compensate for PDGFRb signaling in vSMC/pericytes, in agreement with biochemical analysis.

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