Erika Folestad

Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke

Thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is markedly limited owing to concerns about hemorrhagic complications and the requirement that tPA be administered within 3 h of symptoms. Here we report that tPA activation of latent platelet-derived growth factor-CC (PDGF-CC) may explain these limitations. Intraventricular injection of tPA or active PDGF-CC, in the absence of ischemia, leads to significant increases in cerebrovascular permeability. In contrast, co-injection of neutralizing antibodies to PDGF-CC with tPA blocks this increased permeability, indicating that PDGF-CC is a downstream substrate of tPA within the neurovascular unit. These effects are mediated through activation of PDGF-α receptors (PDGFR-α) on perivascular astrocytes, and treatment of mice with the PDGFR-α antagonist imatinib after ischemic stroke reduces both cerebrovascular permeability and hemorrhagic complications associated with late administration of thrombolytic tPA. These data demonstrate that PDGF signaling regulates blood-brain barrier permeability and suggest potential new strategies for stroke treatment.

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) α and β 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 PDGFRβ ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRα were found in tumor cells, whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ 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-3α and GSK-3β. 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.

Imatinib treatment reduces brain injury in a murine model of traumatic brain injury

Current therapies for Traumatic brain injury (TBI) focus on stabilizing individuals and on preventing further damage from the secondary consequences of TBI. A major complication of TBI is cerebral edema, which can be caused by the loss of blood brain barrier (BBB) integrity. Recent studies in several CNS pathologies have shown that activation of latent platelet derived growth factor-CC (PDGF-CC) within the brain can promote BBB permeability through PDGF receptor α (PDGFRα) signaling, and that blocking this pathway improves outcomes. In this study we examine the efficacy for the treatment of TBI of an FDA approved antagonist of the PDGFRα, Imatinib. Using a murine model we show that Imatinib treatment, begun 45 min after TBI and given twice daily for 5 days, significantly reduces BBB dysfunction. This is associated with significantly reduced lesion size 24 h, 7 days, and 21 days after TBI, reduced cerebral edema, determined from apparent diffusion co-efficient (ADC) measurements, and with the preservation of cognitive function. Finally, analysis of cerebrospinal fluid (CSF) from human TBI patients suggests a possible correlation between high PDGF-CC levels and increased injury severity. Thus, our data suggests a novel strategy for the treatment of TBI with an existing FDA approved antagonist of the PDGFRα.

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

Significance Emerging evidence suggest that the cellular composition of tumors is highly heterogeneous. Subclonal species of malignant cells may account for variability in therapeutic responses and for relapse following treatments. However, little is known about the molecular drivers of specific subsets of cancer cells. Herein, we identify expression of platelet-derived growth factor receptor beta (PDGFRβ) as a previously unrecognized feature of a minor malignant cell population in pancreatic neuroendocrine tumors. By the use of mice genetically deficient for Pdgfd, we reveal a crucial and nonredundant function for signaling by platelet-derived growth factor (PDGF)-DD in promoting functional tumor heterogeneity by providing growth-stimulatory cues. Taken together, the use of drugs targeting PDGFRβ signaling, such as the approved targeted therapy sunitinib, may affect the functional intratumoral cross talk in pancreatic neuroendocrine tumors. 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.

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 β, 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 subpopulations 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.

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.

Neuropilin 1 binds PDGF-D and is a co-receptor in PDGF-D–PDGFRβ signaling

ABSTRACT Platelet-derived growth factor (PDGF)-D is a PDGF receptor β (PDGFRβ)-specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological function remains incompletely understood. In this study, we demonstrate that PDGF-D binds directly to neuropilin 1 (NRP1), in a manner that requires the PDGF-D C-terminal Arg residue. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRβ–NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell–cell junctions in endothelial cells, independently of PDGFRβ, altering the availability of NRP1 for VEGF-A–VEGFR2 signaling. PDGF-D showed differential effects on pericyte behavior in ex vivo sprouting assays compared to PDGF-B. Furthermore, PDGF-D-induced PDGFRβ–NRP1 interaction can occur in trans between molecules located in different cells (endothelial cells and pericytes). In summary, we show that NRP1 can act as a co-receptor for PDGF-D–PDGFRβ signaling and is possibly implicated in intercellular communication in the vascular wall. Highlighted Article: Neuropilin 1 is a co-receptor for PDGF-D, but not PDGF-B, in PDGFRβ signaling. This may represent a new and important aspect in the biology of NRP1 and PDGF-D.

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.

Plasma cholesterol lowering in an AngII‑infused atherosclerotic mouse model with moderate hypercholesterolemia

Atherosclerosis is the main underlying causes of cardiovascular disease. There is a well‑established association between high blood cholesterol levels and the extent of atherosclerosis. Furthermore, atherosclerosis has been proposed to augment abdominal aortic aneurysm (AAA) formation. As patients with AAA often have parallel atherosclerotic disease and are therefore often on cholesterol‑lowering therapy, it is not possible to fully address the independent effects of plasma cholesterol lowering (PCL) treatment on AAA. The present study investigated the effect of angiotensin II (AngII)‑infusion in modestly hypercholesterolemic Ldlr‑/‑Apob100/100Mttpflox/floxMx1‑Cre mice with or without PCL treatment on a morphological and molecular level, in terms of atherosclerosis and AAA development. AngII infusion in the study mice resulted in an increased atherosclerotic lesion area and increased infiltration of inflammatory leukocytes, which was not observed in mice with PCL induced prior to AngII infusion. This suggested that AngII infusion in this mouse model induced atherosclerosis development, and that plasma cholesterol levels represent a controlling factor. Furthermore, AngII infusion in Ldlr‑/‑Apob100/100Mttpflox/floxMx1‑Cre mice caused a modest aneurysmal phenotype, and no differences in AAA development were observed between the different study groups. However, the fact that modest hypercholesterolemic mice did not develop AAA in a classical aneurysmal model indicated that plasma cholesterol levels are important for disease development.

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.