PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands have been expressed under the same heart specific alpha-myosin heavy chain promoter. Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfrα in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFRα agonists supports a role for endogenous PDGFRα agonists in the pathogenesis of cardiac fibrosis.

Tesaglitazar, a dual PPAR-α/γ agonist, hamster carcinogenicity, investigative animal and clinical studies.

Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.

Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development.

During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor beta (PDGFRbeta) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.

Platelet-derived growth factor B retention is essential for development of normal structure and function of conduit vessels and capillaries.

OBJECTIVE: Extracellular retention of PDGF-B has been proposed to play an important role in PDGF-B signalling. We used the PDGF-B retention motif knockout mouse (RetKO) to study the effects of retention motif deletion on development of micro- and macrovascular structure and function. METHODS: Passive and active properties of conduit vessels were studied using myograph techniques and histological examination. Capillary structure and function was studied using measurements of capillary density in skeletal muscle and by assessing aerobic physical performance in a treadmill setup. Cardiac function was assessed using echocardiography. RESULTS: Myograph experiments revealed an increased diameter and stiffness of the aorta in RetKO. Histological examination showed increased media collagen content and a decreased number of aortic wall layers, however with a similar number of vascular smooth muscle cells. This outward eutrophic remodelling of the aorta was accompanied by endothelial dysfunction. RetKO showed decreased capillary density in skeletal muscle and signs of a defective delivery of capillary oxygen to skeletal muscle, as shown by a decreased physical performance. In RetKO mice, echocardiography revealed an adaptive eccentric cardiac hypertrophy. CONCLUSION: We conclude that retention of PDGF-B during development is essential for a normal conduit vessel function in the adult mouse. Furthermore, PDGF-B retention is also necessary for the development of an adequate capillary density, and thereby for a normal oxygen delivery to skeletal muscle. The lack of primary effects on cardiac function supports the redundant role of PDGF-B in cardiac development.

Platelet-derived growth factor receptor-beta promotes early endothelial cell differentiation.

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular stability by promoting the recruitment of PDGF receptor-beta-expressing perivascular cells. Here we present data indicating that early hematopoietic/endothelial (hemangio) precursors express PDGFR-beta based on coexpression with CD31, vascular endothelial growth factor receptor-2, and CD41 in 2 models: mouse yolk sac (embryonic day 8 [E8]) and differentiating mouse embryonic stem cells (embryoid bodies). Expression of PDGFR-beta on hemangioprecursor cells in the embryoid bodies gradually disappeared, and, at E14, expression appeared on perivascular cells. Activation of the PDGFR-beta on the hemangioprecursors accelerated the differentiation of endothelial cells, whereas differentiation of the hematopoietic lineage was suppressed. In E9.5 yolk sacs derived from recombinant mice expressing kinase-active PDGFR-beta with an aspartic acid to asparagine (D894N) replacement in the kinase activating loop and from mice with ubiquitous expression of PDGF-BB driven by the Rosa26 locus, the number of CD41-expressing early hematopoietic cells decreased by 36% and 34%, respectively, compared with staged wild-type littermates. Moreover, enhanced vascular remodeling was evident in the Rosa26-PDGF-BB yolk sacs. We conclude that PDGFR-beta is expressed on early hemangioprecursor cells, regulating vascular/hematopoietic development.

Pericytes limit tumor cell metastasis.

Previously we observed that neural cell adhesion molecule (NCAM) deficiency in beta tumor cells facilitates metastasis into distant organs and local lymph nodes. Here, we show that NCAM-deficient beta cell tumors grew leaky blood vessels with perturbed pericyte-endothelial cell-cell interactions and deficient perivascular deposition of ECM components. Conversely, tumor cell expression of NCAM in a fibrosarcoma model (T241) improved pericyte recruitment and increased perivascular deposition of ECM molecules. Together, these findings suggest that NCAM may limit tumor cell metastasis by stabilizing the microvessel wall. To directly address whether pericyte dysfunction increases the metastatic potential of solid tumors, we studied beta cell tumorigenesis in primary pericyte-deficient Pdgfb(ret/ret) mice. This resulted in beta tumor cell metastases in distant organs and local lymph nodes, demonstrating a role for pericytes in limiting tumor cell metastasis. These data support a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.

Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly.

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

Role of pericytes in vascular morphogenesis.

Pericytes are solitary, smooth muscle-like mural cells that invest the wall of microvessels. For a long time, the functional significance of the presence and distribution of pericytes in the microvasculature was unclear. However, in recent years, the application of experimental genetics to the PDGF-B/PDGFRbeta signaling pathway in mice has provided a range of mutants with primary defects in pericytes, allowing for studies of the physiological consequences of pericyte deficiency in developmental angiogenesis and adult physiology. Interestingly, some of the phenotypic consequences of these mutations resemble human diseases, such as diabetic retinopathy. The studies have also led to the discovery of critical mechanisms involved in pericyte recruitment and differentiation. The present review focuses on genetic data suggesting that pericytes take active part in developmental angiogenic processes.

Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors.

Tumor-infiltrating blood vessels deviate morphologically and biochemically from normal vessels, raising the prospect of selective pharmacological targeting. Current antiangiogenic approaches focus mainly on endothelial cells, but recent data imply that targeting pericytes may provide additional benefits. Further development of these concepts will require deeper insight into mechanisms of pericyte recruitment and function in tumors. Here, we applied genetic tools to decipher the function of PDGF-B and PDGF-Rbeta in pericyte recruitment in a mouse fibrosarcoma model. In tumors transplanted into PDGF-B retention motif-deficient (pdgf-b(ret/ret)) mice, pericytes were fewer and were partially detached from the vessel wall, coinciding with increased tumor vessel diameter and hemorrhaging. Transgenic PDGF-B expression in tumor cells was able to increase the pericyte density in both WT and pdgf-b(ret/ret) mice but failed to correct the pericyte detachment in pdgf-b(ret/ret) mice. Coinjection of exogenous pericytes and tumor cells showed that pericytes require PDGF-Rbeta for recruitment to tumor vessels, whereas endothelial PDGF-B retention is indispensable for proper integration of pericytes in the vessel wall. Our data support the notion that pericytes serve an important function in tumor vessels and highlight PDGF-B and PDGF-Rbeta as promising molecular targets for therapeutic intervention.

Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall.

Several platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) family members display C-terminal protein motifs that confer retention of the secreted factors within the pericellular space. To address the role of PDGF-B retention in vivo, we deleted the retention motif by gene targeting in mice. This resulted in defective investment of pericytes in the microvessel wall and delayed formation of the renal glomerulus mesangium. Long-term effects of lack of PDGF-B retention included severe retinal deterioration, glomerulosclerosis, and proteinuria. We conclude that retention of PDGF-B in microvessels is essential for proper recruitment and organization of pericytes and for renal and retinal function in adult mice.