Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma

Myofibroblast accumulation, subepithelial fibrosis, and vascular remodeling are complicating features of chronic asthma, but the mechanisms are not clear. Platelet-derived growth factors (PDGFs) regulate the fate and function of various mesenchymal cells and have been implicated as mediators of lung fibrosis. However, it is not known whether PDGF-BB signaling via PDGFRβ, which is critical for the recruitment of pericytes to blood vessels, plays a role in airway remodeling in chronic asthma. In the present study, we used a selective PDGFRβ inhibitor (CP-673451) to investigate the role of PDGFRβ signaling in the development of airway remodeling and lung dysfunction in an established mouse model of house dust mite-induced chronic allergic asthma. Unexpectedly, we found that pharmacological inhibition of PDGFRβ signaling in the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and airway smooth muscle thickening. Further studies revealed that the inflammatory response to aeroallergen challenge in mice was associated with decreased PDGF-BB expression and the loss of pericytes from the airway microvasculature. In parallel, cells positive for pericyte markers accumulated in the subepithelial region of chronically inflamed airways. This process was exacerbated in animals treated with CP-673451. The results indicate that perturbed PDGF-BB/PDGFRβ signaling and pericyte accumulation in the airway wall may contribute to airway remodeling in chronic allergic asthma

Studies of the novel PDGFs, focusing on PDGF-D

Thirty years ago the classical platelet-derived growth factors (PDG17s), PDGF-A and PDGF-B were discovered. For a long time they were thought to be the only PDG17 isoforms to exist, but recently two novel PDGF molecules were identified, namely PDGF-C and PDGF-D. This finding was unexpected and indicates that the PDGF signalling system is much more complex than was previously thought. The four PDG17 chains form five different di sulphide-1 inked homo- and heterodimers; PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, which exert their biological effects by binding to, and signalling through two receptor tyrosine kinases, PDGFR-alpha and PDGFR-beta. The biological effects of PDGF-A and PDGF-B have been extensively studied. They are known to be major mitogens for several cell types of mesenchymal origin and have been implicated in several pathological conditions, such as atherosclerosis and fibrotic diseases. The discovery of two new members of the PDG17 family makes it important to further characterize the players in the PDGFR signalling system in order to understand their different biological functions. Part of this work describes the identification of the most recent found member of the PDGFfamily, PDGFD. In a computer-assisted search, a cDNA clone encoding PDGF-D was found. The full-length cDNA encoded a polypeptide of 370 amino acids. The secreted protein turned out to be a disulphide-linked homodimer, which displays a two-domain structure similar to that found in PDGF-C, with an N-terminal CUB domain and a C-terminal PDGF/VEGF homology domain. Secreted PDGF-DD was found to be a latent protein, which requires proteolytic removal of the CUB domains for activity. Proteolytically processed PDGF-DD preferentially binds to and signal through PDGFR-beta. Further, the human genes for PDGF-C and PDGF-D were found to be located on chromosomes 4q32, and 1 1q22.3 to 23.2, respectively. Characterization of the genes showed that the PDGFC gene contained 6 exons, while the PDGFD gene contained 7 exons. The biological role(s) and the in vivo responses of the novel PDG17s are less well known. Data presented herein suggest that PDGF-D might have a role in cardiac fibrosis. This was indicated when PDGF-D was overexpressed in the heart of transgenic mice. Overexpression of PDGF-D induced cardiac fibrosis and hypertrophy, which subsequently caused cardiac failure. In addition, vascular changes, with dilated microvessels, and proliferation of the smooth muscle cells leading to thickening of arterial walls, was seen. The protease uPA has recently been reported to activate PDGF-DD, and in a study where skeletal muscle injury was experimentally induced in uPA deficient mice, uPA was found to be required for efficient regeneration of damaged muscle. Data presented herein establish a role of PDGF-D in skeletal muscle development and regeneration. Using cultured myoblasts, PDGF-DD was found to stimulate proliferation and migration of the myoblasts in vitro, as well as inhibiting their differentiation. In summary, the findings presented in this thesis improve our understanding of the biological function of PDGF-D and enhance the knowledge of the complexity of the PDGF/PDGFR signalling syste

PDGF-C and PDGF-D signaling in vascular diseases and animal models

Members of the platelet-derived growth factor (PDGF) family are well known to be involved in different pathological conditions. The cellular and molecular mechanisms induced by the PDGF signaling have been well studied. Nevertheless, there is much more to discover about their functions and some important questions to be answered. This review summarizes the known roles of two of the PDGFs, PDGF-C and PDGF-D, in vascular diseases. There are clear implications for these growth factors in several vascular diseases, such as atherosclerosis and stroke. The PDGF receptors are broadly expressed in the cardiovascular system in cells such as fibroblasts, smooth muscle cells and pericytes. Altered expression of the receptors and the ligands have been found in various cardiovascular diseases and current studies have shown important implications of PDGF-C and PDGF-D signaling in fibrosis, neovascularization, atherosclerosis and restenosis. (C) 2018 The Authors. Published by Elsevier Ltd

Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor beta, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd(-/-)), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in sub-populations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd(-/-) mice. Furthermore, Pdgfd(-/-) mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis

Extracellular retention of PDGF-B directs vascular remodeling in mouse hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a lethal condition, and current vasodilator therapy has limited effect. Antiproliferative strategies targeting platelet-derived growth factor (PDGF) receptors, such as imatinib, have generated promising results in animal studies. Imatinib is, however, a nonspecific tyrosine kinase inhibitor and has in clinical studies caused unacceptable adverse events. Further studies are needed on the role of PDGF signaling in PH. Here, mice expressing a variant of PDGF-B with no retention motif (Pdgfbret/ret), resulting in defective binding to extracellular matrix, were studied. Following 4 wk of hypoxia, right ventricular systolic pressure, right ventricular hypertrophy, and vascular remodeling were examined. Pdgfbret/ret mice did not develop PH, as assessed by hemodynamic parameters. Hypoxia did, however, induce vascular remodeling in Pdgfbret/ret mice; but unlike the situation in controls where the remodeling led to an increased concentric muscularization of arteries, the vascular remodeling in Pdgfbret/ret mice was characterized by a diffuse muscularization, in which cells expressing smooth muscle cell markers were found in the interalveolar septa detached from the normally muscularized intra-acinar vessels. Additionally, fewer NG2-positive perivascular cells were found in Pdgfbret/ret lungs, and mRNA analyses showed significantly increased levels of Il6 following hypoxia, a known promigratory factor for pericytes. No differences in proliferation were detected at 4 wk. This study emphasizes the importance of extracellular matrix-growth factor interactions and adds to previous knowledge of PDGF-B in PH pathobiology. In summary, Pdgfbret/ret mice have unaltered hemodynamic parameters following chronic hypoxia, possibly secondary to a disorganized vascular muscularization

Distinct Effects of Ligand-Induced PDGFR alpha and PDGFR beta Signaling in the Human Rhabdomyosarcoma Tumor Cell and Stroma Cell Compartments

Platelet-derived growth factor receptors (PDGFR) alpha and beta have been suggested as potential targets for treatment of rhabdomyosarcoma, the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes, with PDGF-D as the solely overexpressed PDGFR beta ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFR alpha were found in tumor cells, whereas PDGFR beta was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFR alpha signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFR beta signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFR beta in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems, where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation, vessel density, and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3 alpha and GSK-3 beta. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation, senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. Cancer Res; 73(7); 2139-49. (C)2013 AACR

Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD.

Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis

<i>Pdgfd</i> is highly expressed in the cardiac vasculature, particularly in arterial bifurcations.

<p>Analysis of heart, whole mounts and sections from wildtype and <i>Pdgfd</i>^+/- mice. (A) Postnatal (day P4) whole mount heart showing X-gal staining (blue) (B) Adult whole mount hearts from 16 weeks old mice <i>Pdgfd</i>^+/+ and <i>Pdgfd</i>^+/- mice showing X-gal staining. (C) Magnification from (B). (D-G) Representative images of sections from heart showing X-gal staining. (D) <i>Pdgfd</i>^+/+, control. (E) Overview of <i>Pdgfd</i>^+/- heart. (F) Higher magnification of <i>Pdgfd</i>^+/- heart, strong staining in blood vessel bifurcation (arrow). (G) <i>Pdgfd</i>^+/- counterstained with hematoxylin and eosin. (H-K) Representative images of <i>Pdgfd</i>^+/- heart sections from 21 weeks old mice, showing podocalyxin (H, J) staining as a marker of endothelial cells (green) and X-gal staining (blue). (H) Artery showing X-gal co-staining with podocalyxin (upper arrow) and X-gal staining outside of podocalyxin staining (lower arrow) and (I) in the same section, artery showing X-gal without podocalyxin. (J) A vein showing limited X-gal staining (arrow), and (K) the same section, without podocalyxin staining. (L-O) Representative confocal images of a <i>Pdgfd</i>^<i>+/-</i> heart section from 16 weeks old mice showing immunofluorescent alpha-smooth muscle actin (αSMA) and enzymatic X-gal staining visualized by transmitted light. (L) Arteries showing αSMA (red) and X-gal staining (black). (M-O) Magnifications from (L), arrows pointing at black X-gal staining. (M) Arteries showing αSMA (red) and X-gal staining (black). (N) αSMA (white), no transmitted light. (O) X-gal staining (black). Scale bars 100 μm.</p

Clinical chemistry markers in serum.

<p>Clinical chemistry markers in serum.</p