Design of substrates and inhibitors of G protein-coupled receptor kinase 2 (GRK2) based on its phosphorylation reaction.

The G protein-coupled receptor kinase (GRK) family consists of seven cytosolic serine/threonine (Ser/Thr) protein kinases, and among them, GRK2 is involved in the regulation of an enormous range of both G protein-coupled receptors (GPCRs) and non-GPCR substrates that participate in or regulate many critical cellular processes. GRK2 dysfunction is associated with multiple diseases, including cancers, brain diseases, cardiovascular and metabolic diseases, and therefore GRK2-specific substrates/inhibitors are needed not only for studies of GRK2-mediated cellular functions but also for GRK2-targeted drug development. Here, we first review the structure, regulation and functions of GRK2, and its synthetic substrates and inhibitors. We then highlight recent work on synthetic peptide substrates/inhibitors as promising tools for fundamental studies of the physiological functions of GRK2, and as candidates for applications in clinical diagnostics.

A high-affinity peptide substrate for G protein-coupled receptor kinase 2 (GRK2).

We synthesized a previously identified ß-tubulin-derived G protein-coupled receptor kinase 2 (GKR2) peptide (GR-11-1; DEMEFTEAESNMN) and its amino-terminal extension (GR-11-1-N; GEGMDEMEFTEAESNMN) and carboxyl-terminal extension (GR-11-1-C; DEMEFTEAESNMNDLVSEYQ) peptides with the aim of finding a high-affinity peptide substrate for GRK2. GR-11-1-C showed high affinity for GRK2, but very low affinity for GKR5. Its specificity and sensitivity for GKR2 were greater than those of GR-11-1 and GR-11-1-N. These findings should be useful in designing tools for probing GKR2-mediated intracellular signaling pathways, as well as GRK2-specific drugs.

Enhanced Osseointegration Capability of Poly(ether ether ketone) via Combined Phosphate and Calcium Surface-Functionalization.

Biomedical applications of poly(ether ether ketone) (PEEK) are hindered by its inherent bioinertness and lack of osseointegration capability. In the present study, to enhance osteogenic activity and, hence, the osseointegration capability of PEEK, we proposed a strategy of combined phosphate and calcium surface-functionalization, in which ozone-gas treatment and wet chemistry were used for introduction of hydroxyl groups and modification of phosphate and/or calcium, respectively. Surface functionalization significantly elevated the surface hydrophilicity without changing the surface roughness or topography. The cell study demonstrated that immobilization of phosphate or calcium increased the osteogenesis of rat mesenchymal stem cells compared with bare PEEK, including cell proliferation, alkaline phosphatase activity, and bone-like nodule formation. Interestingly, further enhancement was observed for samples co-immobilized with phosphate and calcium. Furthermore, in the animal study, phosphate and calcium co-functionalized PEEK demonstrated significantly enhanced osseointegration, as revealed by a greater direct bone-to-implant contact ratio and bond strength between the bone and implant than unfunctionalized and phosphate-functionalized PEEK, which paves the way for the orthopedic and dental application of PEEK.

Increased hepatic inflammation in a normal-weight mouse after long-term high-fat diet feeding.

Among five C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks, one mouse showed a body weight (BW) similar to normal diet (ND)-fed mice. We compared obesity-related parameters of three groups (ND-fed mice, one HFD-fed normal-weight mouse, and HFD-fed overweight mice), including visceral fat weight, serum levels of total cholesterol (TC), glucose, and aminotransferases (AST and ALT), adipocyte size, percentage of crown-like structures, severity of hepatic steatosis, and number of inflammatory foci. Compared to ND-fed mice, the HFD-fed normal-weight mouse exhibited a similar visceral fat weight, similar serum levels of glucose and aminotransferases, and a similar percentage of crown-like structures. On the other hand, the serum TC level, adipocyte size, and hepatic steatosis severity of the HFD-fed normal-weight mouse were intermediate between those of ND-fed mice and HFD-fed overweight mice. Interestingly, the number of hepatic inflammatory foci in the HFD-fed normal-weight mouse was remarkably increased compared with those in HFD-fed overweight mice. These results suggest that having BW or serum ALT levels within normal ranges may not guarantee absence of hepatic inflammation and that the HFD-fed normal-weight mouse can be used as an animal model for the study of liver inflammation, particularly in patients with normal BWs and/or serum ALT values.

Liver cell-targeted delivery of therapeutic molecules.

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2).

A series of amino acid substitutions was made in a previously identified ß-tubulin-derived GRK2 substrate peptide (404DEMEFTEAESNMN416) to examine the role of amino acid residues surrounding the phosphorylation site. Anionic amino acid residues surrounding the phosphorylation site played an important role in the affinity for GRK2. Compared to the original peptide, a modified peptide (Ac-EEMEFSEAEANMN-NH2) exhibited markedly higher affinity for GRK2, but very low affinity for GRK5, suggesting that it can be a sensitive and selective peptide for GRK2.

A superhydrophilic titanium implant functionalized by ozone gas modulates bone marrow cell and macrophage responses.

Bone-forming cells and Mϕ play key roles in bone tissue repair. In this study, we prepared a superhydrophilic titanium implant functionalized by ozone gas to modulate osteoconductivity and inhibit inflammatory response towards titanium implants. After 24 h of ozone gas treatment, the water contact angle of the titanium surface became zero. XPS analysis revealed that hydroxyl groups were greatly increased, but carbon contaminants were largely decreased 24 h after ozone gas functionalization. Also, ozone gas functionalization did not alter titanium surface topography. Superhydrophilic titanium (O3-Ti) largely increased the aspect ratio, size and perimeter of cells when compared with untreated titanium (unTi). In addition, O3-Ti facilitated rat bone marrow derived MSCs differentiation and mineralization evidenced by greater ALP activity and bone-like nodule formation. Interestingly, O3-Ti did not affect RAW264.7 Mϕ proliferation. However, naive RAW264.7 Mϕ cultured on unTi produced a two-fold larger amount of TNFα than that on O3-Ti. Furthermore, O3-Ti greatly mitigated proinflammatory cytokine production, including TNFα and IL-6 from LSP-stimulated RAW264.7 Mϕ. These results demonstrated that a superhydrophilic titanium prepared by simple ozone gas functionalization successfully increased MSCs proliferation and differentiation, and mitigated proinflammatory cytokine production from both naive and LPS-stimulated Mϕ. This superhydrophilic surface would be useful as an endosseous implantable biomaterials and as a biomaterial for implantation into other tissues.

Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is frequently used to monitor phosphorylated peptides or protein kinase activities. However, few reports have compared a radioactivity assay with MALDI-TOF-MS analysis. We analyzed the phosphorylation ratios of 23 peptide substrates for G protein-coupled receptor kinase 2 (GRK2) with different lengths and numbers of negatively charged amino acids by MALDI-TOF-MS. We then examined the correlations between the phosphorylation ratios determined by MALDI-TOF-MS and the radioactivity levels (counts per minute, CPM) determined using a radioactive assay. Using MALDI-TOF-MS, the phosphorylation ratios were greater in the negative mode than in the positive mode. The phosphorylation ratio measured in the negative mode was strongly correlated with the CPM (r = 0.86). The number of acidic amino acids was related to the phosphorylation of peptide substrates by GRK2 (r = 0.53 and 0.46 for the phosphorylation ratio and CPM, respectively). These results suggest that MALDI-TOF-MS is an alternative to radioactive assays for monitoring phosphorylated peptides.

Fabrication of porous calcite using chopped nylon fiber and its evaluation using rats.

Although porous calcite has attracted attention as bone substitutes, limited studies have been made so far. In the present study, porous calcite block was fabricated by introducing chopped nylon fiber as porogen. Ca(OH)2 powder containing 10 wt% chopped nylon fiber was compacted at 150 MPa, and sintered to burn out the fiber and to carbonate the Ca(OH)2 under stream of 1:2 O2-CO2. Sintering of Ca(OH)2 at 750 °C or lower temperature resulted in incomplete burning out of the fiber whereas sintering at 800 °C or higher temperature resulted in the formation of CaO due to the thermal decomposition of Ca(OH)2. However, sintering at 770 °C resulted in complete burning out of the fiber and complete carbonation of Ca(OH)2 to calcite without forming CaO. Macro- and micro-porosities of the porous calcite were approximately 23 and 16%, respectively. Diameter of the macropores was approximately 100 µm which is suitable for bone tissue penetration. Porous calcite block fabricated by this method exhibited good tissue response when implanted in the bone defect in femur of 12-weeks-old rat. Four weeks after implantation, bone bonded on the surface of calcite. Furthermore, bone tissue penetrated interior to the macropore at 8 weeks. These results demonstrated the good potential value of porous calcite as artificial bone substitutes.

Fluorescent polyion complex nanoparticle that incorporates an internal standard for quantitative analysis of protein kinase activity.

We demonstrate a polyion complex (PIC) nanoparticle that contains both a responsive fluorophore and an "internal standard" fluorophore for quantitative measurement of protein kinase (PK) activity. The PK-responsive fluorophore becomes more fluorescent with PK-catalyzed phosphorylation of substrate peptides incorporated in the PIC, while fluorescence from the internal standard remains unchanged during phosphorylation. This new concept will be useful for quantitative PK assays and the discovery of PK inhibitors.

Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase.

High levels of serum phosphate are associated with calcification of human smooth muscle cells (HSMCs). We investigated whether inhibition of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] can reduce inorganic phosphate (Pi)-induced HSMC calcification. Inhibition of PKA or p38 MAPK by inhibitors or small interfering RNAs (siRNAs) reduced Ca levels and alkaline phosphatase activities in HSMCs treated with high Pi, but inhibition of ERK1/2 and JNK showed no significant changes. Moreover, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, and MEK1/2), but JNK inhibitor slightly reduced cell viability. These results show that PKA and p38 MAPK are involved in the Pi-induced calcification of HSMCs, and may be good targets for reducing vascular calcification.

Vascular calcification and cellular signaling pathways as potential therapeutic targets.

Increased vascular calcification (VC) is observed in patients with cardiovascular diseases such as atherosclerosis, diabetes, and chronic kidney disease. VC is divided into three types according to its location: intimal, medial, and valvular. Various cellular signaling pathways are associated with VC, including the Wnt, mitogen-activated protein kinase, phosphatidylinositol-3 kinase/Akt, cyclic nucleotide-dependent protein kinase, protein kinase C, calcium/calmodulin-dependent kinase II, adenosine monophosphate-activated protein kinase/mammalian target of rapamycin, Ras homologous GTPase, apoptosis, Notch, and cytokine signaling pathways. In this review, we discuss the literature concerning the key cellular signaling pathways associated with VC and their role as potential therapeutic targets. Inhibitors to these pathways represent good candidates for use as potential therapeutic agents for the prevention and treatment of VC.

Releasable, Immune-Instructive, Bioinspired Multilayer Coating Resists Implant-Induced Fibrosis while Accelerating Tissue Repair.

Implantable biomaterials trigger foreign body reactions (FBRs), which reduces the functional life of medical devices and prevents effective tissue regeneration. Although existing therapeutic approaches can circumvent collagen-rich fibrotic encapsulation secondary to FBRs, they disrupt native tissue repair. Herein, a new surface engineering strategy in which an apoptotic-mimetic, immunomodulatory, phosphatidylserine liposome (PSL) is released from an implant coating to induce the formation of a macrophage phenotype that mitigates FBRs and improves tissue healing is described. PSL-multilayers constructed on implant surfaces via the layer-by-layer method release PSLs over a 1-month period. In rat muscles, poly(etheretherketone) (PEEK), a nondegradable polymer implant model, induces FBRs with dense fibrotic scarring under an aberrant cellular profile that recruits high levels of inflammatory infiltrates, foreign body giant cells (FBGCs), scar-forming myofibroblasts, and inflammatory M1-like macrophages but negligible amounts of anti-inflammatory M2-like phenotypes. However, the PSL-multilayer coating markedly diminishes these detrimental signatures by shifting the macrophage phenotype. Unlike other therapeutics, PSL-multilayered coatings also stimulate muscle regeneration. This study demonstrates that PSL-multilayered coatings are effective in eliminating FBRs and promoting regeneration, hence offering potent and broad applications for implantable biomaterials.

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging.

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (∼10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.

Biodistribution of (125)I-labeled polymeric vaccine carriers after subcutaneous injection.

Polymeric nanoparticles (NPs) comprised of hydrophilic poly(γ-glutamic acid) in the main chain and hydrophobic phenylalanine in the side chain (γ-PGA-Phe) are a promising vaccine carrier for various kinds of diseases. However, little is known about the fate of subcutaneously administered γ-PGA-Phe NPs. Therefore, we newly synthesized γ-PGA graft phenylalanine and tyrosine conjugates (γ-PGA-Phe-Tyr), and then γ-PGA-Phe-Tyr NPs were labeled with (125)I for monitoring their biodistribution (γ-PGA-Phe-Tyr((125)I) NPs). Dynamic light scattering (DLS) measurements showed that γ-PGA-Phe-Tyr((125)I) NPs showed 200nm in diameter and a negative ζ-potential, which was comparable to those of their precursors. γ-scintigraphic images showed that in mice, subcutaneously injected γ-PGA-Phe-Tyr((125)I) NPs were mainly observed at the site of injection (SOI), but not other organs 1h after administration. However, γ-PGA-PheTyr((125)I) NPs were almost undetectable at the SOI and other organs at 11days postinjection. Similar results were observed when γ-PGA-Phe-Tyr((125)I) NPs were subcutaneously injected into rats. Furthermore, at 11days postinjection, 73±3% of the injected dose of γ-PGA-Phe-Tyr((125)I) NPs was detected in the feces (14±1%) and urine (59±1%). These results clearly showed that subcutaneously injected γ-PGA-Phe-Tyr((125)I) NPs were cleared from the body, and γ-PGA-Phe NPs were safe and effective vaccine carriers.

Bisphenol A (BPA) and Cardiovascular or Cardiometabolic Diseases.

Bisphenol A (BPA; 4,4'-isopropylidenediphenol) is a well-known endocrine disruptor. Most human exposure to BPA occurs through the consumption of BPA-contaminated foods. Cardiovascular or cardiometabolic diseases such as diabetes, obesity, hypertension, acute kidney disease, chronic kidney disease, and heart failure are the leading causes of death worldwide. Positive associations have been reported between blood or urinary BPA levels and cardiovascular or cardiometabolic diseases. BPA also induces disorders or dysfunctions in the tissues associated with these diseases through various cell signaling pathways. This review highlights the literature elucidating the relationship between BPA and various cardiovascular or cardiometabolic diseases and the potential mechanisms underlying BPA-mediated disorders or dysfunctions in tissues such as blood vessels, skeletal muscle, adipose tissue, liver, pancreas, kidney, and heart that are associated with these diseases.

Gene carrier showing all-or-none response to cancer cell signaling.

In this work we designed a novel nano carrier, a linear polyethylenimine (LPEI)-peptide conjugate, for cancer-specific expression of transgenes. The conjugate was easily synthesized by using a click chemistry scheme orthogonal to the reactive side groups of the peptide, which is the substrate of protein kinase Cα (PKCα). Polyplexes of the conjugates with plasmid DNA (pDNA) were intact and stably dispersed even in the presence of cell lysate. Despite this stability, the polyplexes readily dissociated upon phosphorylation of the grafted peptides by PKCα. Because of its endosomal escape ability and adequate susceptibility to PKCα, the polyplexes showed an all-or-none type response to PKCα activity in transgene expression in vitro. The polyplexes achieved cancer tissue-specific transgene expression even for a tumor with a relatively low PKCα activity. Thus the LPEI-peptide conjugate has high potential as a nanocarrier for cancer-targeted gene therapy.

Development of human hepatocellular carcinoma cell-targeted protein cages.

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.

Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex.

Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH(2)) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.

Phosphatidylserine liposome multilayers mediate the M1-to-M2 macrophage polarization to enhance bone tissue regeneration.

An appropriate immune microenvironment, governed by macrophages, is essential for rapid tissue regeneration after biomaterial implantation. The macrophage phenotypes, M1 (inflammatory) and M2 (anti-inflammatory/healing), exert opposing effects on the repair of various tissues. In this study, a new strategy to promote tissue repair and tissue-to-biomaterial integration by M1-to-M2 macrophage transition using artificial apoptotic cell mimetics (phosphatidylserine liposomes; PSLs) was developed using bone as a model tissue. Titanium was also selected as a model substrate material because it is widely used for dental and orthopedic implants. Titanium implants were functionalized with multilayers via layer-by-layer assembly of cationic protamine and negatively charged PSLs that were chemically stabilized to prevent disruption of lipid bilayers. Samples carrying PSL multilayers could drive M1-type macrophages into M2-biased phenotypes, resulting in a dramatic change in macrophage secretion for tissue regeneration. In a rat femur implantation model, the PSL-multilayer-coated implant displayed augmented de novo bone formation and bone-to-implant integration, associated with an increased M1-to-M2-like phenotypic transition. This triggered the proper generation and activation of bone-forming osteoblasts and bone-resorbing osteoclasts relative to their uncoated counterparts. This study demonstrates the benefit of local M1-to-M2 macrophage polarization induced by PSL-multilayers constructed on implants for potent bone regeneration and bone-to-implant integration. The results of this study may help in the design of new immunomodulatory biomaterials. STATEMENT OF SIGNIFICANCE: Effective strategies for tissue regeneration are essential in the clinical practice. The macrophage phenotypes, M1 (inflammatory) and M2 (anti-inflammatory/healing), exert opposing effects on the repair of various tissues. Artificially produced phosphatidylserine-containing liposomes (PSLs) can induce M2 macrophage polarization by mimicking the inverted plasma membranes of apoptotic cells. This study demonstrates the advantages of local M1-to-M2 macrophage polarization induced by PSL-multilayers constructed on implants for effective bone regeneration and osseointegration (bone-to-implant integration). Mechanistically, M2 macrophages promote osteogenesis but inhibit osteoclastogenesis, and M1 macrophages vice versa. We believe that our study makes a significant contribution to the design of new immunomodulatory biomaterials for regenerative medicine because it is the first to validate the benefit of PSLs for tissue regeneration.