Pharmacological inhibition of PDGF-C/neuropilin-1 interaction: A novel strategy to reduce melanoma metastatic potential.

Activation of neuropilin-1 (NRP-1) by platelet derived growth factor (PDGF)-C sustains melanoma invasiveness. Therefore, in the search of novel agents capable of reducing melanoma spreading, PDGF-C/NRP-1 interaction was investigated as a potential druggable target. Since the PDGF-C region involved in NRP-1 binding is not yet known, based on the sequence and structural homology between PDGF-C and vascular endothelial growth factor-A (VEGF-A), we hypothesized that the NRP-1 b1 domain region involved in the interaction with VEGF-A might also be required for PDGF-C binding. Hence, this region was selected from the protein crystal structure and used as target in the molecular docking procedure. In the following virtual screening, compounds from a DrugBank database were used as query ligands to identify agents potentially capable of disrupting NRP-1/PDGF-C interaction. Among the top 45 candidates with the highest affinity, five drugs were selected based on the safety profile, lack of hormonal effects, and current availability in the market: the antipsychotic pimozide, antidiabetic gliclazide, antiallergic cromolyn sodium, anticancer tyrosine kinase inhibitor entrectinib, and antihistamine azelastine. Analysis of drug influence on PDGF-C in vitro binding to NRP-1 and PDGF-C induced migration of human melanoma cells expressing NRP-1, indicated gliclazide and entrectinib as the most specific agents that were active at clinically achievable and non-toxic concentrations. Both drugs also reverted PDGF-C ability to stimulate extracellular matrix invasion by melanoma cells resistant to BRAF inhibitors. The inhibitory effect on tumor cell motility involved a decrease of p130Cas phosphorylation, a signal transduction pathway activated by PDGF-C-mediated stimulation of NRP-1.

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions.

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)ß controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRß is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

PDGF-C promotes cell proliferation partially via downregulating BOP1.

Platelet-derived growth factor C (PDGF-C) is a member of PDGF/VEGF family, which is well-known for important functions in the vascular system. It is widely reported that PDGF-C is able to modulate cell proliferation. However, it is still not very clear about this cell modulating mechanism at the molecular level. In a screening of factors regulated by PDGF-C protein, we fished out a factor called block of proliferation 1 (BOP1), which is a pivotal regulator of ribosome biogenesis and cell proliferation. In this study, we investigated the regulation of BOP1 by PDGF-C and its role in modulating cell proliferation. We found that BOP1 was downregulated at both mRNA and protein levels in cells treated with PDGF-C-containing conditioned medium. On the other hand, BOP1 was upregulated in PDGF-C deficient mice. Furthermore, we confirmed that overexpression of BOP1 inhibited HEK293A cell proliferation, whereas knockdown of BOP1 promoted cell proliferation. The mitogenic effect of PDGF-C could be attenuated by downregulation of BOP1. Our results demonstrate a clear PDGF-C-BOP1 signaling that modulates cell proliferation.

Reversible promoter demethylation of PDGFD confers gemcitabine resistance through STAT3 activation and RRM1 upregulation.

Drug resistance is a major problem in cancer treatment with traditional or targeted therapeutics. Gemcitabine is approved for several human cancers and the first line treatment for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, gemcitabine resistance frequently occurs and is a major problem in successful treatments of these cancers and the mechanism of gemcitabine resistance remains largely unknown. In this study, we identified 65 genes that had reversible methylation changes in their promoters in gemcitabine resistant PDAC cells using whole genome Reduced Representation Bisulfite Sequencing analyses. One of these genes, PDGFD, was further studied in detail for its reversible epigenetic regulation in expression and shown to contribute to gemcitabine resistance in vitro and in vivo via stimulating STAT3 signaling in both autocrine and paracrine manners to upregulate RRM1 expression. Analyses of TCGA datasets showed that PDGFD positively associates with poor outcome of PDAC patients. Together, we conclude that the reversible epigenetic upregulation plays an important role in gemcitabine resistance development and targeting PDGFD signaling alleviates gemcitabine resistance for PDAC treatment.

Thermodynamic Evolution of a Metamorphic Protein: A Theoretical-Computational Study of Human Lymphotactin.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

lncRNA PITPNA-AS1 promotes cell proliferation and metastasis in hepatocellular carcinoma by upregulating PDGFD.

Hepatocellular carcinoma (HCC) ranks high in morbidity and mortality among notorious malignancies because of the lack of effective biomarkers and treatments. LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) plays an oncogenic role in HCC, yet the mechanistic basis remains unprobed. Here using Bioinformatics and PCR analyses, we validated that PITPNA-AS1 expression was significantly increased in HCC. The levels of PITPNA-AS1 in tumors were reversely correlated with the prognosis in HCC patients. Downregulation of PITPNA-AS1 inhibited malignant activities of HCC cells. Next, we elucidated that PITPNA-AS1 acts as a competing endogenous RNA (ceRNA) to sponge miR-363-5p, thereby regulating the expression of platelet-derived growth factor-D (PDGFD). Moreover, the suppression of HCC cell activities by PITPNA-AS1 downregulation can be removed by PDGFD overexpression or miR-363-5p inhibition. Collectively, our work reveals the involvement of the PITPNA-AS1/miR-363-5p/PDGFD regulatory axis in HCC progression.

PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B.

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their ß1 and ß3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-ß (PDGF Receptor ß) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-ß transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

E3 ligase HUWE1 promotes PDGF D-mediated osteoblastic differentiation of mesenchymal stem cells by effecting polyubiquitination of ß-PDGFR.

Mesenchymal stem cells (MSCs) are adult stem cell populations and exhibit great potential in regenerative medicine and oncology. Platelet-derived growth factors (PDGFs) are well known to regulate MSC biology through their chemotactic and mitogenic properties. However, their direct roles in the regulation of MSC lineage commitment are unclear. Here, we show that PDGF D promotes the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into osteoblasts and inhibits hBMSC differentiation into adipocytes. We demonstrate that PDGF D-induced ß-actin expression and polymerization are essential for mediating this differential regulation of osteoblastogenesis and adipogenesis. Interestingly, we found that PDGF D induces massive upward molecular weight shifts of its cognate receptor, PDGF receptor beta (ß-PDGFR) in hBMSCs, which was not observed in fibroblasts. Proteomic analysis indicated that the E3 ubiquitin ligase HECT, UBA, and WWE domain-containing protein 1 (HUWE1) associates with the PDGF D-activated ß-PDGFR signaling complex in hBMSCs, resulting in ß-PDGFR polyubiquitination. In contrast to the well-known role of ubiquitin in protein degradation, we provide evidence that HUWE1-mediated ß-PDGFR polyubiquitination delays ß-PDGFR internalization and degradation, thereby prolonging AKT signaling. Finally, we demonstrate that HUWE1-regulated ß-PDGFR signaling is essential for osteoblastic differentiation of hBMSCs, while being dispensable for PDGF D-induced hBMSC migration and proliferation as well as PDGF D-mediated inhibition of hBMSC differentiation into adipocytes. Taken together, our findings provide novel insights into the molecular mechanism by which PDGF D regulates the commitment of hBMSCs into the osteoblastic lineage.

V-CARMA: A tool for the detection and modification of antigen-specific T cells.

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Downregulation of PDGF-D Inhibits Proliferation and Invasion in Breast Cancer MDA-MB-231 Cells.

BACKGROUND: The platelet derived growth factor-D (PDGF-D) plays an important role in breast tumor aggressiveness. However, limited study has investigated the effect of silencing PDGF-D on the biological function of breast cancer. The purpose of this study is to clarify the potential value of PDGF-D as a target for breast cancer treatment. METHODS: Reverse transcription-polymerase chain reaction and western blot were used to detect PDGF-D expression in 5 different breast cancer cells. The lentiviral vector was usd to silence PDGF-D in MDA-MB-231 cells. Then, Methyl Thiazolyl Tetrazolium was used to detect cell viability, 5-Ethynyl-2'- deoxyuridine and a soft agar assay were used to detect cell proliferation and clonality. Additionally, cell apoptosis after PDGF-D knockdown was measured by Annexin V/ Prodium Iodide staining, and cell migration was detected by trans-well assay. Survival rate and tumor size were measured by nude mice transplantation. RESULTS: The MDA-MB-231 and SK-BR-3 cell lines showed higher PDGF-D expression than the MCF7 cell lines (P<.05). After the PDGF-D gene was silenced, the growth and colony forming abilitys ignificantly decreased (P<.05) together with the induction of apoptosis in MDA-MB-231 cells (P<.05). Moreover, MDA-MB-231 cells with PDGF-D silencing showed significantly diminished aggressive migration and invasion potential compared to other cells (P<.05). In vivo experiments also indicated that PDGF-D silencing inhibited tumor growth and improved the survival rate of tumor-bearing mice. CONCLUSION: Downregulation of PDGF-D had dramatic effects on breast cancer cell proliferation, apoptosis and migration, which indicates that it plays an important role in breast cancer development and progression.

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development.

Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12-18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.

Effect of Omeprazole on Osteoblasts and Osteoclasts in vivo and in the in vitro Model Using Fish Scales.

Omeprazole suppresses excessive secretion of gastric acid via irreversible inhibition of H+/K+-ATPase in the gastric parietal cells. Recent meta-analysis of data revealed an association between the use of proton pump inhibitors (PPIs) and increased risk of bone fractures, but the underlying molecular mechanism of PPI action remains unclear. In this study, we demonstrated that omeprazole directly influences bone metabolism using a unique in vitro bioassay system with teleost scales, as well as the in vivo model. The in vitro study showed that omeprazole significantly increased the activities of alkaline phosphatase and tartrate-resistant acid phosphatase after 6 h of incubation with this PPI. Expression of mRNAs for several osteoclastic markers was upregulated after 3-h incubation of fish scales with 10-7 M omeprazole. The in vivo experiments revealed that the plasma calcium levels significantly increased in the omeprazole-treated group. The results of in vitro and in vivo studies suggest that omeprazole affects bone cells by increasing bone resorption by upregulating expression of osteoclastic genes and promoting calcium release to the circulation. The suggested in vitro bioassay in fish scales is a practical model that can be used to study the effects of drugs on bone metabolism.

Expression and function of PDGF-C in development and stem cells.

Platelet-derived growth factor C (PDGF-C) is a relatively new member of the PDGF family, discovered nearly 20 years after the finding of platelet-derived growth factor A (PDGF-A) and platelet-derived growth factor B (PDGF-B). PDGF-C is generally expressed in most organs and cell types. Studies from the past 20 years have demonstrated critical roles of PDGF-C in numerous biological, physiological and pathological processes, such as development, angiogenesis, tumour growth, tissue remodelling, wound healing, atherosclerosis, fibrosis, stem/progenitor cell regulation and metabolism. Understanding PDGF-C expression and activities thus will be of great importance to various research disciplines. In this review, however, we mainly discuss the expression and functions of PDGF-C and its receptors in development and stem cells.

A Transcriptional Signature of IL-2 Expanded Natural Killer Cells Predicts More Favorable Prognosis in Bladder Cancer.

Activation of natural killer (NK) cell function is regulated by cytokines, such as IL-2, and secreted factors upregulated in the tumor microenvironment, such as platelet-derived growth factor D (PDGF-DD). In order to elucidate a clinical role for these important regulators of NK cell function in antitumor immunity, we generated transcriptional signatures representing resting, IL-2-expanded, and PDGF-DD-activated, NK cell phenotypes and established their abundance in The Cancer Genome Atlas bladder cancer (BLCA) dataset using CIBERSORT. The IL-2-expanded NK cell phenotype was the most abundant in low and high grades of BLCA tumors and was associated with improved prognosis. In contrast, PDGFD expression was associated with numerous cancer hallmark pathways in BLCA tumors compared with normal bladder tissue, and a high tumor abundance of PDGFD transcripts and the PDGF-DD-activated NK cell phenotype were associated with a poor BLCA prognosis. Finally, high tumor expression of transcripts encoding the activating NK cell receptors, KLRK1 and the CD160-TNFRSF14 receptor-ligand pair, was strongly correlated with the IL-2-expanded NK cell phenotype and improved BLCA prognosis. The transcriptional parameters we describe may be optimized to improve BLCA patient prognosis and risk stratification in the clinic and potentially provide gene targets of therapeutic significance for enhancing NK cell antitumor immunity in BLCA.

A Transcriptional Signature of PDGF-DD Activated Natural Killer Cells Predicts More Favorable Prognosis in Low-Grade Glioma.

The binding of platelet-derived growth factor D (PDGF-DD) to the NKp44 receptor activates a distinct transcriptional program in primary IL-2 expanded human natural killer (NK) cells. We were interested in knowing if the PDGF-DD-NKp44 pathway of NK cell activation might play a clinically relevant role in anti-tumor immunity. In order to address this question, we determined transcriptional signatures unique to resting, IL-2 expanded, and PDGF-DD activated, NK cells, in addition to different T cell subsets, and established the abundance of these immune cell phenotypes in The Cancer Genome Atlas (TCGA) low-grade glioma (LGG) dataset using CIBERSORT. Our results show that LGG patient tumors enriched for either the PDGF-DD activated NK cell or memory CD8+ T cell phenotypes are associated with a more favorable prognosis. Combined cell phenotype analyses revealed that patients with LGG tumors enriched for the PDGF-DD activated NK cell phenotype and the CD4+ T helper cell phenotype had a more favorable prognosis. High expression of transcripts encoding members of the killer cell lectin-like receptor (KLR) family, such as KLRK1 and KLRC2, KLRC3 and KLRC4 in LGG tumors were associated with more favorable prognosis, suggesting that these NK cell family receptors may play a prominent role in LGG anti-tumor immunity. Finally, many of the TCGA findings were reciprocated in LGG patients from the Chinese Glioma Genome Atlas (CGGA) dataset. Our results provide transcriptomic evidence that PDGF-DD activated NK cells and KLR family receptors may play an important clinical role in immune surveillance of LGG.

Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture.

In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios.

The HIF1α-PDGFD-PDGFRα axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin.

BACKGROUND: Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies. METHODS: Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence. RESULTS: HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway. CONCLUSIONS: HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.

Assessing the Performance of Traveling-salesman based Automated Path Searching (TAPS) on Complex Biomolecular Systems.

Though crucial for understanding the function of large biomolecular systems, locating the minimum free energy paths (MFEPs) between their key conformational states is far from trivial due to their high-dimensional nature. Most existing path-searching methods require a static collective variable space as input, encoding intuition or prior knowledge of the transition mechanism. Such information is, however, hardly available a priori and expensive to validate. To alleviate this issue, we have previously introduced a Traveling-salesman based Automated Path Searching method (TAPS) and demonstrated its efficiency on simple peptide systems. Having implemented a parallel version of this method, here we assess the performance of TAPS on three realistic systems (tens to hundreds of residues) in explicit solvents. We show that TAPS successfully located the MFEP for the ground/excited state transition of the T4 lysozyme L99A variant, consistent with previous findings. TAPS also helped identifying the important role of the two polar contacts in directing the loop-in/loop-out transition of the mitogen-activated protein kinase kinase (MEK1), which explained previous mutant experiments. Remarkably, at a minimal cost of 126 ns sampling, TAPS revealed that the Ltn40/Ltn10 transition of lymphotactin needs no complete unfolding/refolding of its ß-sheets and that five polar contacts are sufficient to stabilize the various partially unfolded intermediates along the MFEP. These results present TAPS as a general and promising tool for studying the functional dynamics of complex biomolecular systems.

Diet-regulated production of PDGFcc by macrophages controls energy storage.

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.

PDGF-D activation by macrophage-derived uPA promotes AngII-induced cardiac remodeling in obese mice.

Obesity-induced secretory disorder of adipose tissue-derived factors is important for cardiac damage. However, whether platelet-derived growth factor-D (PDGF-D), a newly identified adipokine, regulates cardiac remodeling in angiotensin II (AngII)-infused obese mice is unclear. Here, we found obesity induced PDGF-D expression in adipose tissue as well as more severe cardiac remodeling compared with control lean mice after AngII infusion. Adipocyte-specific PDGF-D knockout attenuated hypertensive cardiac remodeling in obese mice. Consistently, adipocyte-specific PDGF-D overexpression transgenic mice (PA-Tg) showed exacerbated cardiac remodeling after AngII infusion without high-fat diet treatment. Mechanistic studies indicated that AngII-stimulated macrophages produce urokinase plasminogen activator (uPA) that activates PDGF-D by splicing full-length PDGF-D into the active PDGF-DD. Moreover, bone marrow-specific uPA knockdown decreased active PDGF-DD levels in the heart and improved cardiac remodeling in HFD hypertensive mice. Together, our data provide for the first time a new interaction pattern between macrophage and adipocyte: that macrophage-derived uPA activates adipocyte-secreted PDGF-D, which finally accelerates AngII-induced cardiac remodeling in obese mice.