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.

Structural insights into the modulation of PDGF/PDGFR-ß complexation by hyaluronan derivatives.

Angiogenesis is an important physiological process playing a crucial role in wound healing and cancer progression. Vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are key players in angiogenesis. Based on previous findings regarding the modulation of VEGF activity by glycosaminoglycans (GAG), here we explore the interaction of hyaluronan (HA)-based GAG with PDGF and its receptor PDGFR-ß by applying molecular modeling and dynamics simulations in combination with surface plasmon resonance (SPR). Computational analysis on the interaction of oligo-hyaluronan derivatives with different sulfation pattern and functionalization shows that these GAG interact with PDGF in relevant regions for receptor recognition, and that high sulfation as well as modification with the TAMRA group convey stronger binding. On the other hand, the studied oligo-hyaluronan derivatives are predicted to scarcely recognize PDGFR-ß. SPR results are in line with the computational predictions regarding the binding pattern of HA tetrasaccharide (HA4) derivatives to PDGF and PDGFR-ß. Furthermore, our experimental results also show that the complexation of PDGF to PDGFR-ß can be modulated by HA4 derivatives. The results found open the path for considering HA4 derivatives as potential candidates to be exploited for modulation of the PDGF/PDGFR-ß signaling system in angiogenesis and related disease conditions.

PDGF-BB/SA/Dex injectable hydrogels accelerate BMSC-mediated functional full thickness skin wound repair by promoting angiogenesis.

Wound healing is a well-orchestrated dynamic and interactive process, which needs a favorable microenvironment and suitable angiogenesis. Platelet derived growth factor-BB (PDGF-BB) plays a crucial role in wound healing. However, the short half-life of PDGF-BB limits its efficacy. In the present study, we successfully synthesized an injectable hydrogel with sodium alginate (SA) and dextran (Dex) as a delivery system to simultaneously deliver PDGF-BB and bone marrow-derived mesenchymal stem cells (BMSCs) in the wound. Our work demonstrates that the PDGF-BB protein enhanced the survival, migration and endothelial cell (EC) differentiation of BMSCs in vitro. The PDGF-BB/SA/Dex hydrogels could sustainably release PDGF-BB with excellent biocompatibility in vitro and in vivo. Besides, these composite hydrogels loaded with BMSCs could accelerate wound healing by improving epithelialization and collagen deposition. In addition, the PDGF-BB/SA/Dex hydrogels promoted the EC-differentiation of transplanted BMSCs and proliferation of hair follicle stem cells in the wound. Furthermore, the expressions of angiogenesis-specific markers, PDGFR-ß, p-PI3K, p-Akt, and p-eNOS, were obviously increased in the PDGF-BB/SA/Dex/BMSCs group. In conclusion, the PDGF-BB/SA/Dex injectable hydrogels could accelerate BMSC-mediated skin wound healing by promoting angiogenesis via the activation of the PDGF-BB/PDGFR-ß-mediated PI3K/Akt/eNOS pathway, which may provide a new therapeutic strategy for stem cell therapy in wound healing.

Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Rare variant analysis of 4241 pulmonary arterial hypertension cases from an international consortium implicates FBLN2, PDGFD, and rare de novo variants in PAH.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined. METHODS: To identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource - Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD. RESULTS: Seven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×, p = 2.5e-5). At least eight novel pediatric candidate genes carrying de novo variants have plausible roles in lung/heart development. CONCLUSIONS: Rare variant analysis of a large international consortium identified two new candidate genes-FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling. Trio analysis predicted that ~ 15% of pediatric IPAH may be explained by de novo variants.

Ligand-Linked Association-Dissociation in Transport Proteins and Hormone Receptors.

Ligand-linked changes in the aggregation state of biological macromolecules occur and have importance in several physiological processes, e.g., the response of hormone receptors, cooperative ligand binding, and others. The mathematical formalisms that express the thermodynamics governing these processes are complex, as they are required to describe observations made under experimental conditions in which many parameters may be simultaneously varied. The description of the functional behaviour of proteins that present ligand-linked association-dissociation events must accommodate cases where both the binding stoichiometries and reaction mechanisms are variable. In this paper, we review some paradigmatic cases that cover different structural arrangements and binding modes, with special attention to the case of dissociating homodimeric transport proteins and receptors. Even though we cannot pretend to be comprehensive on the proteins presenting this behaviour, we believe that we can attempt to be comprehensive on the structural arrangements and thermodynamic properties of these systems, which fall into a limited set of possible types.

Aptamer-Functionalized Three-Dimensional Carbon Nanowebs for Ultrasensitive and Free-Standing PDGF Biosensor.

Research on flexible biosensors is mostly focused on their use in obtaining information on physical signals (such as temperature, heart rate, pH, and intraocular pressure). Consequently, there are hardly any studies on using flexible electronics for detecting biomolecules and biomarkers that cause diseases. In this study, we propose a flexible, three-dimensional carbon nanoweb (3DCNW)-based aptamer sensor to detect the platelet-induced growth factor (PDGF), which is an oncogenic biomarker. As a template for the 3D structure, poly(acrylonitrile) (PAN) nanowebs were synthesized using a facile electrospinning process. The PAN nanowebs were then subjected to chemical vapor deposition with copper powder. This was followed by Cu etching to generate carbon protrusions on the web surface. As an active site, PDGF-B binding aptamer was introduced on the 3DCNW surface to form biosensor electrodes. The 3DCNW-based aptasensor exhibited excellent sensitivity (down to 1.78 fM), with high selectivity, reversibility, and stability to PDGF-BB.

Cord blood-derived platelet concentrates as starting material for new therapeutic blood components prepared in a public cord blood bank: from product development to clinical application.

BACKGROUND: There are many advantages to using cord blood (CB) as a source of therapeutic platelet and plasma derivatives for regenerative medicine. These include availability, universal use, young donor source, and virally safe biological material, rich in tissue regenerative factors. MATERIALS AND METHODS: We aimed to validate a bioprocess design for the production of cord blood-derived platelet concentrates (CBPC) in a public Cord Blood Bank (CBB). CBPC was defined as a product of 10±5 mL, 1,000±200×109/L total platelets, free of erythrocytes and leukocytes. A total of 300 CB units were centrifuged in two steps to enrich for platelets, in compliance with Good Manufacturing Practice. The samples were tested for the degree of platelet activation present, and the levels of growth factor were analysed to evaluate their potential function. CBPC were then activated after thawing with 10% calcium gluconate to generate platelet gels (CBPG) to treat patients with diabetic foot ulcers. RESULTS: After processing, 84% of the products fulfilled the acceptance criteria. Final products contained 1,017±149×106 platelets/mL in 10±3mL of plasma. Platelet recovery was 50±9%. The methods described here ensure depletion of white and red blood cells down to a residual concentration of 0.2±0.1×106/mL and 0.03±0.02×106/mL, respectively. Platelets showed low levels of activation during processing, but were significantly activated after thawing, as indicated by an increase in CD62p expression. The growth factors EGF, VEGF, bFGF, PDGF AB/BB and TGF-ß1 were at concentrations of 1,706±123 pg/mL; 1,602±227 pg/mL; 314±26 pg/mL; 30±1.5 ng/mL; 24±2 ng/mL (mean±standard error of mean), respectively. For clinical evaluation, a total of 21 CBPG were applied in 3 patients, with no reported adverse events and improvement of ulcers in all of them. DISCUSSION: We designed and validated a highly reproducible, closed system method to manufacture high quality CBPC suitable for clinical applications using CB units not suitable for transplantation in a public CBB.

Long-term local PDGF delivery using porous microspheres modified with heparin for tendon healing of rotator cuff tendinitis in a rabbit model.

In this study, we prepared the platelet-derived growth factor-containing porous microspheres modified with heparin (PDGF/Hep-PMSs) and investigated their anti-inflammatory and tendon healing effects on rotator cuff (RC) tendinitis rabbit model. PDGF/Hep-PMSs suppressed the mRNA levels of six pro-inflammatory cytokines (i.e., MMP-3, MMP-13, COX-2, ADAMTS-5, IL-6, and TNF-α) in inflamed tenocytes. Long-term local delivery of PDGF/Hep-PMSs into tendon tissues of RC tendinitis decreased the mRNA levels of six pro-inflammatory cytokines and increased the mRNA levels of anti-inflammatory cytokines including IL-4, IL-10, and IL-13. Anti-inflammatory effects of PDGF/Hep-PMSs might have contributed to enhance the collagen content, tenogenic markers, stiffness, and tensile strength of tendons, eventually leading to tendon restoration. Our findings suggest that the long-term local PDGF delivery of PDGF/Hep-PMSs have a great potential to enhance tendon healing of RC tendinitis by suppressing inflammation responses.

Effects of incorporation of granule-lyophilised platelet-rich fibrin into polyvinyl alcohol hydrogel on wound healing.

Dressings are commonly used to treat skin wounds. In this study, we aimed to develop a new scaffold composed of a polyvinyl alcohol (PVA) hydrogel containing granule-lyophilised platelet-rich fibrin (G-L-PRF) as a dressing. G-L-PRF was prepared by freeze-drying and was then incorporated into PVA hydrogel by freezing-thawing. Notably, the mechanical strength and degradation rate of the scaffold were found to be related to G-L-PRF concentrations, reaching 6.451 × 10-2 MPa and 17-22%, respectively, at a concentration of 1%. However, the strength decreased and the degradation was accelerated when the G-L-PRF concentration was over 1%. The elastic properties and biocompatibility of the scaffold were independent of G-L-PRF concentration, and both showed excellent elasticity and biocompatibility. The release of vascular endothelial growth factor and platelet-derived growth factor-AB was no significant time dependent. Additionally, application of 1% G-L-PRF/PVA to acute full-thickness dorsal skin wounds accelerated wound closure at days 7 and 9. Healing also increased on day 11. Histological and immunohistochemical analyses showed that the scaffold enhanced granulation tissue, maturity, collagen deposition, and new vessel formation. These results demonstrated that the prepared G-L-PRF/PVA scaffolds accelerated wound healing in acute full-thickness skin wounds, suggesting potential applications as an ideal wound dressing.

Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears.

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration. Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-ß (PDGFRß) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks. Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRß expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF. In conclusion, PDGF-BB-coated scaffold increased PDGFRß expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears. STATEMENT OF SIGNIFICANCE: Meniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use. We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells. The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties. This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

Preliminary in vitro and in vivo investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor.

Aberrant expression of wild-type and mutant forms of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases has been implicated in various oncologic indications such as leukemias, gliomas, and soft tissue sarcomas. Clinically used kinase inhibitors imatinib and sunitinib are potent inhibitors of wild-type PDGFR family members, but show reduced binding to mutant forms. Here we describe compound 5 which binds to both wild-type and oncogenic mutant forms of PDGFR family members, and demonstrates both cellular and in vivo activity.

Evaluation of the therapeutic effects of rice husk nanosilica combined with platelet-derived growth factor in hepatic veno-occlusive disease.

Veno-occlusive disease is an important pattern of hepatotoxicity associated with antineoplastic drugs. The study investigated the possible therapeutic effects of RHS nanoparticles combined with a PDGF on veno-occlusive disease (VOD) in liver elicited in rats with DAC. In this work, nanosilica (SiO2) was successfully prepared from rice husk, and its physicochemical characteristics were investigated using EDX, XRD, N2 adsorption-desorption isotherm, SEM, and TEM. Forty-eight male Sprague-Dawely rats were distributed into 6 groups, with 8 rats in each. The first group served as the control. In the second group, animals were infused with DAC (0.015 mg/kg; 1-3 days) by intraperitoneal injection (i.p.). In the third group, rats were injected i.p. with DAC, and then at 24 h following the last dose of DAC, received nano-RHS incorporated with PDGF twice a week for 4 weeks. In the fourth group, normal animals were injected with RHS. In the fifth group, normal rats received PDGF, and in the sixth group, normal rats received nano-RHS combined with PDGF. The prepared nanosilica showed type II adsorption isotherm characteristic for mesoporous materials with a specific surface area of 236 m2/g. TEM imaging confirmed the production of nanoparticles via the followed preparation procedure. Radical scavenging potential for nano-RHS was determined using two different in-vitro assays: DPPH, and ABTS radicals. The results of this work show that administration of nano-RHS combined with PDGF significantly reversed the oxidative stress effects of DAC as evidenced by a decrease in liver function. It can be concluded that the nano-RHS combined with PDGF is useful in preventing oxidative stress and hepatic VOD induced by chemotherapy such as DAC.

PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration.

This study aimed to develop a functionally graded membrane (FGM) to prevent infection and promote tissue regeneration. Poly(l-lactide-co-d,l-lactide) encapsulating platelet-derived growth factor (PDLLA-PDGF) or metronidazole (PDLLA-MTZ) was electrospun to form a nanofibrous layer on the inner or outer surface of a clinically available collagen membrane, respectively. The membrane was characterized for the morphology, molecule release profile, in vitro and in vivo biocompatibility, and preclinical efficiency for alveolar ridge regeneration. The PDLLA-MTZ and PDLLA-PDGF nanofibers were 800-900 nm in diameter, and the thicknesses of the functional layers were 20-30 µm, with sustained molecule release over 28 days. All of the membranes tested were compatible with cell survival in vitro and showed good tissue integration with minimal fibrous capsule formation or inflammation. Cell proliferation was especially prominent on the PDLLA-PDGF layer in vivo. On the alveolar ridge, all FGMs reduced wound dehiscence compared with the control collagen membrane, and the FGM with PDLLA-PDGF promoted osteogenesis significantly. In conclusion, the FGMs with PDLLA-PDGF and PDLLA-MTZ showed high biocompatibility and facilitated wound healing compared with conventional membrane, and the FGM with PDLLA-PDGF enhanced alveolar ridge regeneration in vivo. The design represents a beneficial modification, which may be easily adapted for future clinical use.

Quantifying 3D chemotaxis in microfluidic-based chips with step gradients of collagen hydrogel concentrations.

Cell migration is an essential process involved in crucial stages of tissue formation, regeneration or immune function as well as in pathological processes including tumor development or metastasis. During the last few years, the effect of gradients of soluble molecules on cell migration has been widely studied, and complex systems have been used to analyze cell behavior under simultaneous mechano-chemical stimuli. Most of these chemotactic assays have, however, focused on specific substrates in 2D. The aim of the present work is to develop a novel microfluidic-based chip that allows the long-term chemoattractant effect of growth factors (GFs) on 3D cell migration to be studied, while also providing the possibility to analyze the influence of the interface generated between different adjacent hydrogels. Namely, 1.5, 2, 2.5 and 4 mg ml-1 concentrations of collagen type I were alternatively combined with 5, 10 or 50 ng ml-1 concentrations of PDGF and VEGF (as a negative control). To achieve this goal, we have designed a new microfluidic device including three adjacent chambers to introduce hydrogels that allow the generation of a collagen concentration step gradient. This versatile and simple platform was tested by using dermal human fibroblasts embedded in 3D collagen matrices. Images taken over a week were processed to quantify the number of cells in each zone. We found, in terms of cell distribution, that the presence of PDGF, especially in small concentrations, was a strong chemoattractant for dermal human fibroblasts across the gels regardless of their collagen concentration and step gradient direction, whereas the effects of VEGF or collagen step gradient concentrations alone were negligible.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration.

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

Nanoemulsion-Based Hydrogel for Topical Delivery of Highly Skin-Permeable Growth Factor Combinations: Preparation and In Vitro Evaluation.

Topical administration of growth factors has been suggested as a promising strategy for promoting the healing process and skin regeneration in wound management. However, several restrictions hinder their successful clinical use; specifically, limited percutaneous absorption causes inconsistent efficacy, and various growth factors with specific functionalities are required at different stages of healing. To overcome these shortcomings, previously we have constructed highly skin-permeable analogues of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and platelet-derived growth factor-A (PDGF-A) (LMWP-EGF, LMWP-IGF-I and LMWP-PDGF-A) by genetically conjugating the low-molecular-weight protamine (LMWP) to their N-terminus. In the present study, we determined the optimal concentration ratio of these growth factors by investigating In Vitro cell proliferation and the scratch wound repairing assay. After confirming synergetic effects of growth factors in combinations, we developed a topical delivery system consisting of a nanoemulsion (NE)-dispersed polyvinylpyrrolidone hydrogel loaded with all three growth factors. In Vitro permeability studies were also performed to assess whether the LMWP-conjugated growth factors in the formulation enhanced their skin permeation compared to native growth factors. Combinations of native or LMWP-fused growth factors significantly promoted fibroblast proliferation and scratch wound recovery, and the synergy of LMWP-EGF, LMWP-IGF-I and LMWP-PDGF-A was optimal at a ratio of 100:100:10 by concentration. The growth factor combination-loaded NE appeared to be spherical under cryo-transmission electron microscopy and the average droplet diameter was 127±4.30 nm. The LMWP-conjugated growth factors allowed significantly higher skin permeation than native growth factors from the NE-dispersed hydrogel. Thus, the LMWP-conjugated growth factor combination-loaded NE-dispersed hydrogel is expected to induce more rapid and prolonged wound healing.

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy.

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

Analysis of DNA in Phosphate Buffered Saline Using Kinetic Capillary Electrophoresis.

Kinetic capillary electrophoresis (KCE) methods are useful in the study of kinetics and equilibrium properties of interactions between DNA and its binding partners (ligands). KCE experiments are typically performed in a narrow set of "conventional" low-conductivity run buffers while DNA-ligand interactions in biological systems occur in physiological fluids, characterized by high ionic strengths. The nature and ionic strength of the buffer, in which DNA-ligand interaction occurs, can significantly influence the binding. Therefore, KCE experiments meant to study such interactions would greatly benefit if they could be performed in physiological buffers, such as phosphate buffered saline (PBS). No previous KCE studies of DNA used PBS as the run buffer. Here, we test the feasibility of using PBS as a KCE run buffer for analysis of DNA and show that its usage under standard KCE conditions renders DNA undetectable. We uncover the causes of this previously unreported detrimental effect and come up with a modification of KCE which allows one to overcome it. We apply the modified KCE method to an experimental model of a platelet-derived growth factor (PDGF) protein and its DNA aptamer, which was selected in PBS, and show that the results obtained in PBS run buffer are much closer to previously reported values than those which were obtained with a conventional low-conductivity capillary electrophoresis (CE) buffer.

Platelet-derived growth factor receptor/platelet-derived growth factor (PDGFR/PDGF) system is a prognostic and treatment response biomarker with multifarious therapeutic targets in cancers.

Progress in cancer biology has led to an increasing discovery of oncogenic alterations of the platelet-derived growth factor receptors (PDGFRs) in cancers. In addition, their overexpression in numerous cancers invariably makes PDGFRs and platelet-derived growth factors (PDGFs) prognostic and treatment markers in some cancers. The oncologic alterations of the PDGFR/PDGF system affect the extracellular, transmembrane and tyrosine kinase domains as well as the juxtamembrane segment of the receptor. The receptor is also involved in fusions with intracellular proteins and receptor tyrosine kinase. These discoveries undoubtedly make the system an attractive oncologic therapeutic target. This review covers elementary biology of PDGFR/PDGF system and its role as a prognostic and treatment marker in cancers. In addition, the multifarious therapeutic targets of PDGFR/PDGF system are discussed. Great potential exists in the role of PDGFR/PDGF system as a prognostic and treatment marker and for further exploration of its multifarious therapeutic targets in safe and efficacious management of cancer treatments.