Collagen-derived Peptide Research Articles

Preventive Effects of Collagen-Derived Dipeptide Prolyl-Hydroxyproline against Dexamethasone-Induced Muscle Atrophy in Mouse C2C12 Skeletal Myotubes.

Glucocorticoids, commonly used to manage inflammatory diseases, can induce muscle atrophy by accelerating the breakdown of muscle proteins. This research delves into the influence of Prolyl-hydroxyproline (Pro-Hyp), a collagen-derived peptide, on muscle atrophy induced with dexamethasone (DEX), a synthetic glucocorticoid, in mouse C2C12 skeletal myotubes. Exposure to DEX (10 μM) for 6 days resulted in a decrease in myotube diameter, along with elevated mRNA and protein levels of two muscle-atrophy-related ubiquitin ligases, muscle atrophy F-box (MAFbx, also known as atrogin-1) and muscle ring finger 1 (MuRF-1). Remarkably, treatment with 0.1 mM of Pro-Hyp mitigated the reduction in myotube thickness caused by DEX, while promoting the phosphorylation of Akt, mammalian target of rapamycin (mTOR), and forkhead box O3a (Foxo3a). This led to the inhibition of the upregulation of the ubiquitin ligases atrogin-1 and MuRF-1. These findings indicate the potential significance of Pro-Hyp as a promising therapeutic target for countering DEX-induced muscle atrophy.

Evaluation of Uveitis Induced in Rats by a Type I Collagen Peptide as a Model for Childhood Arthritis-associated Uveitis.

Chronic asymptomatic and acute symptomatic anterior uveitis are forms of ocular inflammation associated with juvenile idiopathic arthritis (JIA) Chronic JIA-associated uveitis is characterized by young age of onset, female predilection, oligoarthritis, and antinuclear antibody (ANA) positivity. Acute JIA-associated uveitis predominantly affects older male juveniles who also develop enthesitis. A type I collagen-derived peptide (melanin-associated antigen [MAA]) induces anterior uveitis in rodents. In this study, we evaluated MAA-induced uveitis in rats as a potential model for JIA-uveitis. We characterized MAA-induced uveitis by assessing its relationship to age and sex; tracking the occurrence of arthritis, enthesitis, and ANA positivity; and measuring vitreous fluid inflammatory biomarkers. Juvenile and adult and male and female Lewis rats (Rattus norvegicus) were inoculated with MAA. Slit-lamp biomicroscopy, indirect ophthalmoscopy, and joint examinations were performed 3 times weekly. Rats were euthanized at 4 wk after MAA inoculation, and plasma ANA testing, vitreous inflammatory biomarker assays, and globe histopathology assessments were conducted. Uveitis, arthritis, ANA status, levels of inflammatory biomarkers, histopathology, and joint tomographic images were assessed in relation to age and sex and compared with nonuveitic controls. All MAA-immunized rats developed uveitis characterized by anterior chamber fibrin, iridal vessel dilation, and miosis, and uveal and choroidal lymphocytic infiltration. Levels of the vitreous fluid biomarker CCL5 were higher in uveitic rats compared with control rats. Time to uveitis onset, clinical uveitis scores, and biomarker levels did not differ based on age or sex. None of the MAA-exposed rats had arthritis, enthesitis, or ANA. None of the rats inoculated with MAA that had been treated with matrix metallopeptidase 1 had clinical, histologic, or immunohistochemical evidence of ocular inflammation. In contrast to JIA-associated uveitis in humans, MAA-induced uveitis in rats is not associated with age or sex predilections and MAA is not arthritogenic.

Synthetic peptides activating discoidin domain receptor 2 and collagen-binding integrins cooperate to stimulate osteoblast differentiation of skeletal progenitor cells.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk

The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle. We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types. Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases. This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132).

Anti-angiogenic collagen IV-derived peptide target engagement with αvβ3 and α5β1 in ocular neovascularization models

AXT107, a collagen-derived peptide that binds integrins αvβ3 and α5β1 with high affinity, suppresses vascular endothelial growth factor (VEGF) signaling, promotes angiopoietin 2-induced Tie2 activation, and suppresses neovascularization (NV) and vascular leakage. Immunohistochemical staining for αvβ3 and α5β1 was markedly increased in NV compared with normal retinal vessels. After intravitreous injection of AXT107, there was no staining with an anti-AXT107 antibody on normal vessels but robust staining of NV that co-localized with αvβ3 and α5β1. Likewise, after intravitreous injection, fluorescein amidite-labeled AXT107 co-localized with αvβ3 and α5β1 on NV but not normal vessels. AXT107 also co-localized with αv and α5 at cell-cell junctions of human umbilical vein endothelial cells (HUVECs). AXT107-integrin binding was demonstrated by exvivo cross-linking/pull-down experiments. These data support the hypothesis that AXT107 therapeutic activity is mediated through binding αvβ3 and α5β1 which are markedly upregulated on endothelial cells in NV providing selective targeting of diseased vessels which has therapeutic and safety benefits.

Fibroblast-targeting polymeric nanovehicles to enhance topical wound healing through promotion of PAR-2 receptor-mediated endocytosis.

The level of collagen production critically determines skin wound contraction. If an intelligent skin drug delivery technology that enables collagen production in a specific wound skin area is developed, a breakthrough in wound healing treatment would be expected. However, such an intelligent drug delivery technology has not yet been developed as much as in the field of anticancer therapy. In this study, we propose a smart drug delivery system using polymeric nanovehicles (PNVs), in which the periphery is conjugated with a fibroblast-targeting collagen-derived peptide, KTTKS (Lys-Thr-Thr-Lys-Ser). We showed that surface engineering of PNVs with simultaneous PEGylation and peptide patching improved the dispersibility of PNVs, while promoting selective cellular uptake to fibroblasts via PAR-2 receptor-mediated endocytosis. In vitro collagen production and in vivo wound healing assays revealed that curcumin-loaded fibroblast-targeting PNVs significantly enhanced collagen production and wound healing activities, thus promising effective skin tissue regeneration.

Adhesion and proliferation properties of type I collagen-derived peptide for possible use in skin tissue engineering application.

The surface properties of three-dimensional scaffolds are improved by coating or covalently linking certain adhesion-promoting proteins or peptides. In the present study, the effect of type I collagen-derived peptide (GKNGDDGEA) on adhesion and proliferation of HaCaT keratinocytes and NIH3T3 murine fibroblast cell lines was studied to assess its suitability for possible skin tissue engineering applications. Cell adhesion and proliferation of HaCaT and NIH3T3 were found to be enhanced by peptide coating. The optimum peptide coating densities to obtain the best cell adhesion and proliferation were found to be 0.827 µmoles/cm2 and 0.62 µmoles/cm2 for HaCaT and NIH3T3, respectively. Cell adhesion, in the presence of anti-integrin α1 antibody, inhibited attachment of NIH3T3 cells indicating the involvement of integrin α1 receptor. However, the attachment of HaCaT cells was not affected by anti-integrin treatment. The higher expression of paxillin confirmed the effect of the peptide in mediating focal adhesion kinases (FAKs) in cell adhesion and proliferation. Gene expression analysis was performed on cell migration proteins like Rho, Rac, Cdc42, integrin receptor α1, and β1, and the extracellular matrix modulating proteins like MMP2, TIMP, and COL1A1 to validate their role on the peptide-mediated cell proliferation. Immunofluorescence analysis showed the distribution and localisation of phospho-FAKon cells cultured on the peptide-coated surfaces. Results support the role of peptides in enhancing cell adhesion and proliferation properties.

Collagen-derived peptide, DGEA, inhibits pro-inflammatory macrophages in biofunctional hydrogels

Macrophages are innate immune cells that play important roles in wound healing. Particularly, M1 macrophages are considered pro‐inflammatory and promote initial phases of inflammation. Long-term exposure to inflammatory stimuli causes an increase in M1 macrophages, which contributes to chronic inflammation. Activated M1 macrophages have been shown to upregulate integrin α2β1 expression. To interfere with α2β1 binding, we designed a biofunctional hydrogel utilizing a collagen I-derived peptide, DGEA (Asp-Gly-Glu-Ala). We hypothesize that M1 macrophage activation can be reduced in the presence of DGEA. Effects of DGEA on M1 macrophages were studied via soluble delivery and immobilization within poly(ethylene glycol) (PEG) hydrogels. We demonstrate that M1 macrophage activation is reduced both via soluble delivery of DGEA in 2D and via immobilized DGEA in a 3D PEG-DGEA hydrogel. This novel biomaterial can manipulate inflammatory macrophage activation and can be applied to prevent chronic inflammatory conditions via macrophage manipulation.Graphical abstract

Collagen-Derived Di-Peptide, Prolylhydroxyproline (Pro-Hyp): A New Low Molecular Weight Growth-Initiating Factor for Specific Fibroblasts Associated With Wound Healing.

Many cells and soluble factors are involved in the wound healing process, which can be divided into inflammatory, proliferative, and remodeling phases. Fibroblasts play a crucial role in wound healing, especially during the proliferative phase, and show heterogeneity depending on lineage, tissue distribution, and extent of differentiation. Fibroblasts from tissue stem cells rather than from healthy tissues infiltrate wounds and proliferate. Some fibroblasts in the wound healing site express the mesenchymal stem cell marker, p75NTR. In the cell culture system, fibroblasts attached to collagen fibrils stop growing, even in the presence of protein growth factors, thus mimicking the quiescent nature of fibroblasts in healthy tissues. Fibroblasts in wound healing sites proliferate and are surrounded by collagen fibrils. These facts indicate presence of new growth-initiating factor for fibroblasts attached to collagen fibrils at the wound healing site, where the collagen-derived peptide, prolyl-hydroxyproline (Pro-Hyp), is generated. Pro-Hyp triggers the growth of p75NTR-positive fibroblasts cultured on collagen gel but not p75NTR-negative fibroblasts. Thus, Pro-Hyp is a low molecular weight growth-initiating factor for specific fibroblasts that is involved in the wound healing process. Pro-Hyp is also supplied to tissues by oral administration of gelatin or collagen hydrolysate. Thus, supplementation of gelatin or collagen hydrolysate has therapeutic potential for chronic wounds. Animal studies and human clinical trials have demonstrated that the ingestion of gelatin or collagen hydrolysate enhances the healing of pressure ulcers in animals and humans and improves delayed wound healing in diabetic animals. Therefore, the low molecular weight fibroblast growth-initiating factor, Pro-Hyp, plays a significant role in wound healing and has therapeutic potential for chronic wounds.

Circulating markers of collagen types I, III, and IV in patients with dilated cardiomyopathy: relationships with myocardial collagen expression.

AimsCollagen‐derived peptides such as collagen I C‐terminal telopeptide (CITP) and procollagen III N‐terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression.Methods and resultsIn 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = −0.41, P < 0.001 and r = −0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B‐type natriuretic peptide (r = 0.32, P = 0.003) and γ‐glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end‐diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end‐diastolic volume index nor with ejection fraction. Each collagen‐derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA.ConclusionsOur study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra‐cardiac organ injury in heart failure.

Evaluation of Hydrogels Presenting Extracellular Matrix-Derived Adhesion Peptides and Encapsulating Cardiac Progenitor Cells for Cardiac Repair.

Cell therapy is an emerging paradigm for the treatment of heart disease. In spite of the exciting and promising preclinical results, the benefits of cell therapy for cardiac repair in patients have been modest at best. Biomaterials-based approaches may overcome the barriers of poor differentiation and retention of transplanted cells. In this study, we prepared and tested hydrogels presenting extracellular matrix (ECM)-derived adhesion peptides as delivery vehicles for c-kit+ cardiac progenitor cells (CPCs). We assessed their effects on cell behavior in vitro as well as cardiac repair in rats undergoing ischemia reperfusion. Hydrogels presenting the collagen-derived GFOGER peptide induced cardiomyocyte differentiation of CPCs as demonstrated by increased expression of cardiomyocyte structural proteins. However, conditioned media obtained from GFOGER hydrogels showed lower levels of secreted reparative factors. Interestingly, following injection in rats undergoing ischemia-reperfusion, treatment with CPCs encapsulated in nonadhesive RDG-presenting hydrogels resulted in the preservation of cardiac contractility and attenuation of postinfarct remodeling whereas the adhesion peptide-presenting hydrogels did not induce any functional improvement. Retention of cells was significantly higher when delivered with nonadhesive hydrogels compared to ECM-derived peptide gels. These data suggest that factors including cell differentiation state, paracrine factors and interaction with biomaterials influence the effectiveness of biomaterials-based cell therapy. A holistic consideration of these multiple variables should be included in cell-biomaterial combination therapy designs.

Engineering the intestinal basement membrane microenvironment using PEG-based hydrogels

Event Abstract Back to Event Engineering the intestinal basement membrane microenvironment using PEG-based hydrogels Victor Hernandez-Gordillo1, Gi Hun Choi1, Mollie Smoak2, Rebecca Carrier3 and Linda Griffith1 1 Massachusetts Institute of Technology, Biological Engineering, United States 2 Louisiana State University and Agricultural Center, Department of Biological and Agricultural Engineering, United States 3 Northeastern University, Department of Chemical Engineering, United States Introduction: Intestinal organoids or “mini-guts” are multilobulated structures composed of budding crypts, epithelial cells and well-defined lumens. Mini-guts have gained interest as a tool to study intestinal biology and as a platform for drug screening because they resemble aspects of the intestinal complexity found in vivo[1]. Mini-guts are cultured embedded in ill-defined Matrigel-based hydrogels. Lot-to-lot variability and the presence of residual growth factors in Matrigel can negatively impact the biological outcomes. Thus, the development of a well-defined matrix that can recapitulate aspects of the basement membrane for intestinal organoid culture is of vital importance. Poly(ethylene glycol, PEG)-based hydrogels are good candidates as synthetic matrices. They can be tailored to different applications by varying the stiffness, degradability and cell adhesion properties. Here we report our efforts to develop PEG-based hydrogels functionalized with a fibronectin-derived peptide (SYNK-RGDS) or a collagen-derived peptide (GFOGER) for intestinal organoid culture. Materials and Methods: Hydrogels were prepared by first reacting a 40 KDa, 8-arm PEG-vinyl sulfone with cell adhesive peptides containing single free thiols groups. Then cross-linked using matrix metalloproteinase sensitive peptides harboring two free thiols groups. We incorporated two fibronectin-derived peptides (RGDS and SYNK-RGDS) and one derived from collagen (GFOGER). Single intestinal stem cells were embedded in these hydrogels either directly during hydrogel assembly or indirectly using a two-step encapsulation. The two-step encapsulation allowed us to modulate the stiffness and cell adhesion properties of the bottom and top hydrogel. Results and Discussion: Preliminary results suggest that direct encapsulation of single cells obtained from mouse organoids in these PEG-based hydrogels results in formation of organoids similar to those that form in Matrigel (Fig. 1b). The PEG-GFOGER hydrogel showed higher number of organoids when compared to PEG-Synk-RGDS hydrogel. A two steps encapsulation procedure with varying stiffness and cell adhesion properties resulted in a completely different morphology. Mouse cells developed multicellular growth structures that resemble the villi of the intestine (Fig. 1c). Further analysis will be needed to confirm the identity of these structures. Figure 1. a) Overall strategy to created PEG-Based hydrogels. b-c) Representative confocal image of mouse intestinal stem cells directly embedded b) or two-steps embedding strategy c) after 7 days of culture in PEG-GFOGER hydrogel systems. Actin is depicted in green and nuclei in blue. Conclusions: We were able to identify hydrogel culture conditions that allow single mouse cells to develop into three-dimensional structures that resemble mouse organoids in direct cell encapsulation or multicellular growth that resembles the villi when using two stem cell encapsulation. DARPA; NIH; Boston Children Hospital

Cell adhesion and growth enabled by biomimetic oligopeptide modification of a polydopamine-poly(ethylene oxide) protein repulsive surface.

Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine–poly(ethylene oxide) (PDA–PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm2 and 700 fmol/cm2 for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA–PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses.Electronic supplementary materialThe online version of this article (doi:10.1007/s10856-015-5583-3) contains supplementary material, which is available to authorized users.

세드롤과 콜라겐 유래 펩타이드의 피부노화 개선효과

Skin aging is the phenomena finally expressed on the skin surface and related to the changes in the micro-structure of the skin texture. Which is resulted in wrinkle formation and uneven tone of skin and so on. In this study, the synergy effect of Cedrol and a collagen-derived peptide in type Ⅲ collagen synthesis was evaluated by in vitro test. The physiological skin state of 22 female volunteers was measured after using the cosmetics for 4 weeks. Results showed that Cedrol and a collagen-derived peptide had the excellent synergy effect in type Ⅲ collagen synthesis. The cosmetics improved skin microrelief, star configurations, skin gloss, skin tone, hydration and elasticity except skin lightening. In conclusion, this study proved that Cedrol and collagen-derived peptide had the synergy effect of type Ⅲ collagen synthesis in the cell level and cosmetics with those was improved skin aging in human volunteer test.

Wound healing activity of a collagen-derived cryptic peptide.

Wound healing involves a well-controlled series of interactions among cells and several mediators leading to the restoration of damaged tissue. Degradation of the extracellular matrix (ECM) protein collagen during remodelling of wound tissue leads to the release of bioactive peptides that can possibly influence the healing process. The RGD-containing, antioxidative collagen peptide E1 isolated in an earlier work was screened in this study for its ability to influence multiple steps of the wound healing process. E1 was assayed for and found to be chemotactic. Excision and incision wounds were created on separate groups of rats and E1 was administered topically. The wound tissues were isolated on the 4th and 8th days post-wound and subjected to biochemical and biophysical analysis. A significant decrease in lipid peroxides in the treatment group confirmed the in vivo antioxidant capacity of E1. The treatment group also displayed significant increase in total protein, collagen and amino sugar synthesis indicating faster ECM formation. The significantly increased rate of wound contraction and reepithelialisation along with higher tensile strength of the wound tissue corroborated the results of biochemical analysis. The results confirm the significant role played by collagen peptides in accelerating the healing process and justify their possible use as a pharmaceutical agent.

Endocannabinoids control platelet activation and limit aggregate formation under flow.

BackgroundThe endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.ObjectivesOur aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.Methods and ResultsWe studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.ConclusionsEndocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.

Collagen-Derived Peptide N-Acetylated Proline-Glycine-Proline in Intervetebral Disc Recruits Cartilage Endplate Stem Cells by Binding with CXC Receptor 2

Introduction Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain. But our comprehension about development of the intervertebral disc (IVD) and pathogenesis of disc degeneration is unclear, which is an obstacle for appropriate therapies. Recently, some reports identified stem/progenitor cells within particular IVD niches and cartilage endplate. These cells together with mesenchymal stem cells (MSC) are recruited to nucleus pulposus(NP) and annulus fibrosus(AF) to generate regenerative functions in a healthy IVD. The possibility of stimulating these stem cells holds promise for future therapy for DDD. But factors which recruit stem cells to NP and AF are unknown. There are increasing evidences that N-acetylated proline-glycine-proline (N-AC-PGP), which is a degradation fragment of collagen, plays an important role in inflammatory cell recruitment. But whether it presents in IVD and whether it is a chemokine of stem cell have remained elusive. Materials and Methods In this study, we detected the presence of N-AC-PGP by liquid chromatography-mass spectrum in IVD with different degeneration grade and the enzymes responsible for generating N-AC-PGP, matrix metalloproteases 8 and 9 and prolyl endopeptidase (PE), are also quantified by ELISA in these IVD samples. To found out the role of N-AC-PGP in IVD, cartilage endplate stem cells (CESC) chemotaxis assay was performed by Tanswell assay. Results We demonstrate the correlation between N-AC-PGP content in IVD and content of MMP-8and9 and PE.N-AC-PGP increase with increasing degeneration grade of IVD. Then, we described the generation pathway of N-AC-PGP in IVD. Finally, we demonstrated that N-AC-PGP can recruit CESC by binding with CXC receptors 2 on CESC. Conclusion These findings reveal a possible regeneration mechanism in IVD, which depends on the chemotaxis of N-AC-PGP for CESC and indicate a new therapy by using N-AC-PGP to stimulate stem cells in IVD. Disclosure of Interest None declared References Snelgrove RJ, Jackson PL, Hardison MT, et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science 2010;330(6000):90–94 Gaggar A, Jackson PL, Noerager BD, et al. A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. J Immunol 2008;180(8):5662–5669 Weathington NM, van Houwelingen AH, Noerager BD, et al. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nat Med 2006;12(3):317–323

Collagen-Derived Peptide N-Acetylated Proline-Glycine-Proline in Intervetebral Disc Recruits Cartilage Endplate Stem Cells by Binding with CXC Receptor 2

Introduction Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain. But our comprehension about development of the intervertebral disc (IVD) and pathogenesis of disc degeneration is unclear, which is an obstacle for appropriate therapies. Materials and Methods Recently, some reports identified stem/progenitor cells within particular IVD niches and cartilage endplate. These cells together with mesenchymal stem cells are recruited to nucleus pulposus (NP) and annulus fibrosus (AF) to generate regenerative functions in a healthy IVD. The possibility of stimulating these stem cells holds promise for future therapy for DDD. But factors which recruit stem cells to NP and AF are unknown. There are increasing evidences that N-acetylated proline-glycine-proline (N-AC-PGP), which is a degradation fragment of collagen, plays an important role in inflammatory cell recruitment. But whether it presents in IVD and whether it is a chemokine of stem cell have remained elusive. Results In this study, we demonstrated the presence of N-AC-PGP in IVD and found the correlation between its content in IVD and degeneration grade of IVD. Then we described the generation pathway of N-AC-PGP in IVD. At last, we demonstrated that N-AC-PGP can recruit cartilage endplate stem cells (CESC) by binding with CXC receptors 2 on CESC. Conclusion These findings reveal a possible regeneration mechanism in IVD, which depends on the chemotaxis of N-AC-PGP for CESC and indicate a new therapy by using N-AC-PGP to stimulate stem cells in IVD. Disclosure of Interest None declared

Type ii collagen peptide activates Akt leading to NF-κb up-regulation in osteoarthritic chondrocytes

Type ii collagen peptide activates Akt leading to NF-κb up-regulation in osteoarthritic chondrocytes

Tunable delivery of bioactive peptides from hydroxyapatite biomaterials and allograft bone using variable‐length polyglutamate domains

Hydroxyapatite (HA) biomaterials and allograft bone are common alternatives to autogenous grafts; however, these materials lack the strong osteoinductive potential of autologous bone. Previous studies have established that polyglutamate domains, which bind selectively to HA, can be engineered onto bioactive peptides as a mechanism for coupling osteoinductive signals onto HA and allograft. In the current investigation, we adapted the polyglutamate approach to tailor delivery of a model collagen-derived peptide, Asp-Gly-Glu-Ala (DGEA), by manipulating the number of glutamates in the HA binding domain. Specifically, DGEA was modified with diglutamate (E2-DGEA), tetraglutamate (E4-DGEA), or heptaglutamate (E7-DGEA), and it was found that initial peptide binding to HA and allograft was significantly enhanced as the number of glutamates increased. We also determined that the rate of release of polyglutamate-DGEA from substrates over a 5-day interval increased proportionally as the number of glutamate residues was decreased. Additionally, we tuned the peptide release rate by creating mixtures of E2-DGEA, E4-DGEA, and E7-DGEA, and observed that release kinetics of the mixtures were distinct from pure solutions of each respective peptide. These collective results suggest that variable-length polyglutamate domains provide an effective mechanism for controlled delivery of osteoregenerative peptides on HA-containing bone graft materials.