Primary human endothelial cells secrete agents that reduce responsiveness to lysophosphatidic acid (LPA)

The plasma level of LPA (lysophosphatidic acid) (200–600 nM) is well within the range that promotes proliferation and migration of vascular ECs (endothelial cells), yet vessels are quiescent and stable. In this report, we considered one explanation for this paradox: that ECs secrete agents that attenuate responsiveness to LPA. Indeed, we observed that CM (conditioned medium) from confluent, quiescent cultures of primary HUVECs (human umbilical vein ECs) contained an agent that inhibited LPA-mediated signalling events and cellular responses. The putative inhibitor, which we tentatively call ILMR (inhibitor of LPA-mediated responsiveness) seemed to act on cells (instead of at the level of LPA) by suppressing the ability of LPA receptor 1 to signal. The amount and/or activity of ILMR was regulated by growth factors; exposing HUVECs to VEGF-A (vascular endothelial growth factor A), but not bFGF (basic fibroblast growth factor), reduced the amount and/or activity of ILMR in CM. We conclude that in addition to promoting angiogenesis directly, VEGF-A can also act indirectly by modulating the bioactivity of angiomodulators such as LPA

Diabetes Disrupts the Response of Retinal Endothelial Cells to the Angiomodulator Lysophosphatidic Acid

The objectives of this study were to investigate how diabetes mellitus (DM) influences responsiveness of retinal neovessels to lysophosphatidic acid (LPA) and to elucidate the underlying mechanism. To this end, we used an ex vivo assay in which neovessels sprouted from retinal explants (isolated from either control or DM mice) when cultured between two layers of collagen and in the presence of vascular endothelial growth factor-A. While DM had no effect on the formation of neovessels, it prevented LPA-induced regression. High-glucose (HG) treatment of retinal explants mimicked the DM phenotype. Similarly, primary retinal endothelial cells (RECs), which were subjected to HG treatment, organized into tubes that were resistant to LPA. HG caused LPA resistance within RECs by elevating ROS, which activated Src-family kinases that stimulated the extracellular signal–related kinase (Erk) pathway, which antagonized LPA-mediated signaling events that were required for regression. This ROS/Src/Erk pathway mechanism appeared to be the same route by which DM induced LPA resistance of retinal neovessels. We conclude that DM/HG reprograms signaling pathways in RECs to induce a state of LPA resistance

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types

Retinal Pigment Epithelium and Müller Progenitor Cell Interaction Increase Müller Progenitor Cell Expression of PDGFRα and Ability to Induce Proliferative Vitreoretinopathy in a Rabbit Model

Purpose. Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment characterized by redetachment of the retina as a result of membrane formation and contraction. A variety of retinal cells, including retinal pigment epithelial (RPE) and Müller glia, and growth factors may be responsible. Platelet-derived growth factor receptor alpha (PDGFRα) is found in large quantities in PVR membranes, and is intrinsic to the development of PVR in rabbit models. This study explores the expression of PDGFR in cocultures of RPE and Müller cells over time to examine how these two cell types may collaborate in the development of PVR. We also examine how changes in PDGFRα expression alter Müller cell pathogenicity. Methods. Human MIO-M1 Müller progenitor (MPC) and ARPE19 cells were studied in a transmembrane coculture system. Immunocytochemistry and Western blot were used to look at PDGFRα, PDGFRβ, and GFAP expression. A transfected MPC line cell line expressing the PDGFRα (MIO-M1α) was generated, and tested in a rabbit model for its ability to induce PVR. Results:. The expression of PDGFRα and PDGFRβ was upregulated in MIO-M1 MPCs cocultured with ARPE19 cells; GFAP was slightly decreased. Increased expression of PDGFRα in the MIO-M1 cell line resulted in increased pathogenicity and enhanced ability to induce PVR in a rabbit model. Conclusions:. Müller and RPE cell interaction can lead to upregulation of PDGFRα and increased Müller cell pathogenicity. Müller cells may play a more active role than previously thought in the development of PVR membranes, particularly when stimulated by an RPE-cell-rich environment. Additional studies of human samples and in animal models are warranted

Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

.Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans.<0.001 vs. no atherosclerosis or control group).Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target

PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

Purpose The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing

ER Stress-Induced Aggresome Trafficking of HtrA1 Protects Against 1! Proteotoxicity

High temperature requirement A1 (HtrA1) belongs to an ancient protein family that is linked to various human disorders. The precise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrA1 remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is implicated in unfolded protein response. In highly secretory cells of the retinal pigmented epithelia, endoplasmic reticulum (ER) stress upregulated HtrA1. HtrA1 co-localized with vimentin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions displayed reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mammalian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis

Cambogin Is Preferentially Cytotoxic to Cells Expressing PDGFR

Platelet-derived growth factor receptors (PDGFRs) have been implicated in a wide array of human malignancies, including medulloblastoma (MB), the most common brain tumor of childhood. Although significant progress in MB biology and therapeutics has been achieved during the past decades, MB remains a horrible challenge to the physicians and researchers. Therefore, novel inhibitors targeting PDGFR signaling pathway may offer great promise for the treatment of MB. In the present study, we investigated the cytotoxicity and mechanisms of cambogin in Daoy MB cells. Our results show that cambogin triggers significant S phase cell cycle arrest and apoptosis via down regulation of cyclin A and E, and activation of caspases. More importantly, further mechanistic studies demonstrated that cambogin inhibits PDGFR signaling in Daoy and genetically defined mouse embryo fibroblast (MEF) cell lines. These results suggest that cambogin is preferentially cytotoxic to cells expressing PDGFR. Our findings may provide a novel approach by targeting PDGFR signaling against MB