Platelet-derived Growth factor-AB Research Articles

Response Assessment of 68Ga-DOTA-E-[c(RGDfK)]2 PET/CT in Lung Adenocarcinoma Patients Treated with Nintedanib Plus Docetaxel.

Nintedanib is an oral angiokinase inhibitor used as second-line treatment for non-small cell lung cancer. New radiotracers, such as 68Ga-DOTA-E-[c(RGDfK)]2, that target αvβ3 integrin might have an impact as a noninvasive method for assessing angiogenesis inhibitors. Methods: From July 2011 through October 2015, 38 patients received second-line nintedanib plus docetaxel. All patients underwent PET/CT with 68Ga-DOTA-E-[c(RGDfK)]2 radiotracer and blood-sample tests to quantify angiogenesis factors (fibroblast growth factor, vascular endothelial growth factor, and platelet-derived growth factor AB) before and after completing 2 therapy cycles. Results: Of the 38 patients, 31 had available baseline and follow-up PET/CT. Baseline lung tumor volume addressed with 68Ga-DOTA-E-[c(RGDfK)]2 PET/CT correlated with serum vascular endothelial growth factor levels, whereas baseline lung/liver SUVmax index correlated with platelet-derived growth factor AB. After treatment, the overall response rate and disease control rate were 7.9% and 47.3%, respectively. A greater decrease in lung tumor volume (-37.2% vs. -27.6%) was associated with a better disease control rate in patients (P = 0.005). Median progression-free survival was 3.7 mo. Nonsmokers and patients with a higher baseline lung tumor volume were more likely to have a higher progression-free survival (6.4 vs. 3.74 [P = 0.023] and 6.4 vs. 2.1 [P = 0.003], respectively). Overall survival was not reached. Patients with a greater decrease in lung SUVmax (not reached vs. 7.1 mo; P = 0.016) and a greater decrease in the lung/spleen SUVmax index (not reached vs. 7.1; P = 0.043) were more likely to have a longer overall survival. Conclusion:68Ga-DOTA-E-[c(RGDfK)]2 PET/CT is a potentially useful tool for assessing responses to angiogenesis inhibitors. Further analysis and novel studies are warranted to identify patients who might benefit from this therapy.

Optimization of pure platelet-rich plasma preparation: A comparative study of pure platelet-rich plasma obtained using different centrifugal conditions in a single-donor model.

While it has been proved that centrifugal conditions for pure platelet-rich plasma (P-PRP) preparation influence the cellular composition of P-PRP obtained, the optimal centrifugal conditions to prepare P-PRP have not yet been identified. In the present study, platelet-containing plasma (PCP) was prepared with the first-spin of different double-spin methods and P-PRP was prepared with different double-spin methods. Whole-blood analysis was performed to evaluate the cellular composition of PCP and P-PRP. The basal and ADP-induced CD62P expression rates of platelets were assessed by flow cytometry to evaluate the function of platelets in PCP and P-PRP. Enzyme-linked immune sorbent assay was performed to quantify interleukin-1β, tumor necrosis factor-α, platelet-derived growth factor AB and transforming growth factor β1 concentrations of PCP and P-PRP. Correlations between the cellular characteristics and cytokine concentrations of P-PRP were analyzed by Pearson correlation analysis. Effects of P-PRP on the proliferation, survival and migration of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes were evaluated by a Cell Counting Kit-8 assay, live/dead staining and Transwell assay, respectively. The results showed that centrifugation at 160 × g for 10 min and 250 × g for 15 min successively captured and concentrated platelets and growth factors significantly more efficiently with preservation of platelet function compared with other conditions (P<0.05). The correlation analysis showed that the similar leukocyte concentrations and leukocyte-reducing efficiencies resulted in similar pro-inflammatory cytokine concentrations in P-PRP (P>0.05) and the maximization of platelet concentration, platelet enrichment factor, platelet capture efficiency and platelet function resulted in the maximization of growth factor concentrations in P-PRP obtained using the optimal conditions (P<0.05). Compared with P-PRP obtained under other conditions, P-PRP obtained under the optimal conditions significantly promoted the proliferation and migration of cells (P<0.05) and did not alter cell survival (P>0.05). Therefore, centrifugation at 160 × g for 10 min and 250 × g for 15 min successively with removal of the buffy coat as a crucial step may provide an optimal preparation system of P-PRP for clinical application.

A Method for the Activation of Platelet-Rich Plasma via Bead Mill Homogenizer for Mesenchymal Stem Cell Culture.

Activated platelet-rich plasma (PRP) has been studied as a replacement for fetal bovine serum (FBS) in stem cell culture. However, current methods are time-consuming or require addition of exogenous substances to activate PRP, which have disadvantages in clinical applications. In this study, we developed a new method for PRP activation using a bead mill homogenizer and compared it with previous methods of PRP activation. PRP was prepared via a two-step centrifugation process and activated via calcium (Ca-PRP), freeze-thaw cycles (FT-PRP), or bead mill homogenizer processing (BM-PRP). Quantification of growth factors in PRP revealed that all forms of activated PRP released higher levels of platelet-derived growth factor-AB and transforming growth factor-β1 than those in platelet-poor plasma; however, BM-PRP resulted in significantly higher levels of growth factors than those from Ca-PRP and FT-PRP. Next, we analyzed the ability of the various forms of PRP to stimulate proliferation, migration, and differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Our results showed that BM-PRP significantly increased proliferation and migration rates of UCB-MSCs while maintaining the phenotypical properties and stem cell abilities of MSCs. Therefore, the developed method could be suitable for PRP activation, and the BM-activated PRP could be an adequate replacement for FBS in stem cell culture.

The impact of the centrifuge characteristics and centrifugation protocols on the cells, growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF) clot and membrane

AbstractL-PRF (leukocyte- and platelet-rich fibrin) is one of the four families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The first objective of this article was to evaluate the mechanical vibrations appearing during centrifugation in four models of commercially available table-top centrifuges used to produce L-PRF and the impact of the centrifuge characteristics on the cell and fibrin architecture of a L-PRF clot and membrane. The second objective of this article was to evaluate how changing some parameters of the L-PRF protocol may influence its biological signature, independently from the characteristics of the centrifuge.In the first part, four different commercially available centrifuges were used to produce L-PRF, following the original L-PRF production method (glass-coated plastic tubes, 400 g force, 12 minutes). The tested systems were the original L-PRF centrifuge (Intra-Spin, Intra-Lock, the only CE and FDA cleared system for t...

The association of maternal lymphatic markers and critical congenital heart defects in the fetus-A population based case-control study.

The objective ot this study was to investigate whether lymphatic markers measured in women during the second trimester are associated with critical congenital heart defects (CCHDs) in offspring. This is a retrospective cohort study of pregnant women who participated in the California Prenatal Screening Program. CCHD data in the offspring was captured by linking birth certificate data with hospital patient discharge records. Second trimester samples were assayed for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) AA/BB, and PDGF AB. Logistic models were used to evaluate the association between lymphatic biomarkers and CCHD. Models were adjusted for other serum biomarkers and maternal characteristics. Results are presented in odds ratios (OR) with 95% confidence intervals (CI). We identified 93 cases with CCHDs and 194 controls without CCHDs. The crude and adjusted OR for log (ln) VEGF was 1.07 (95%CI 0.94-1.22) and 1.08 (95%CI 0.94-1.24), respectively; for ln PDGF AB/BB was 0.93 (95%CI 0.6-1.35) and 0.58 (95%CI 0.32-1.05), respectively. There was a significant association between ln PDFG AA and CCHDs (crude OR 1.83 (95%CI 1.05-3.2); adjusted OR 2.41 (95%CI 1.06-5.44)). Levels of circulating PDGF AA were highest in cases with hypoplastic left heart syndrome (HLHS) (mean 8.78 +/- 1.54 pg/ml). In this study, increased mid-pregnancy maternal serum levels of PDGF AA were associated with CCHDs in offspring. The highest PDGF AA levels were found in mothers of fetuses with HLHS. These findings may be useful in screening for CCHDs and offer insight into their association with nuchal translucency.

An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices.

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-β, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression (FN1, SERPINE1, COL1A1, CTGF, MMP7, and ACTA2) increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of HOPX In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

Determination of 16 serum angiogenic factors in stage I non-small cell lung cancer using a bead-based multiplex immunoassay.

The aim of the study was to identify significant abnormalities in angiogenic factor profiles occurring at early-stage non-small cell lung cancer (NSCLC). Contrary to the previous studies, our research included patients with only stage I NSCLC, which allowed for estimating the utility of circulating angiogenic factors in early NSCLC detection. The investigation was performed in serum samples collected from individuals with untreated NSCLC (n=41) and a matched control group of healthy individuals (n=61). All patients had histopathologically-confirmed stage IA or IB NSCLC. Serum concentrations of 16 angiogenesis markers comprising growth factors, receptors, cytokines, chemokines and hormones, were measured using a bead-based multiplex immunoassay. Among the determined proteins, osteopontin, platelet-derived growth factor-AB and -BB (PDGF-AB/BB) and hepatocyte growth factor (HGF) demonstrated the largest increase in concentration in NSCLC patients as compared with the non-cancer group. ROC curve analysis confirmed their high discriminatory abilities in detection of early NSCLC (AUC=0.809, 95%CI 0.725-0.881). Serum levels of the studied angiogenic factors differed slightly between patients with lung adenocarcinoma and squamous cell carcinoma. The study demonstrated that osteopontin, PDGF-AB/BB, and HGF might add up to the methods used in NSCLC diagnosis. Future research should address the question about their specificity and performance in early NSCLC detection in a combination with various putative lung cancer markers.

Platelet-Derived Growth Factor Receptor β But Not α Mediates Cardiac Fibrosis In Vitro and In Vivo

Background: Platelet-derived growth factor (PDGF) is one of the mediators in cardiac fibrosis but its role has not been fully explored. The aim of this study is to investigate the role of PDGF subtypes (AA, AB, BB, & CC) and their receptors (PDGFRα & β) and signalling pathways involved in cardiac fibrosis both in vitro and in vivo. Methods: Cardiac myocyte (NCM) hypertrophy and fibroblast (NCF) collagen synthesis stimulated by PDGF isoforms (10ng/mL) for 48hours, were determined by [3H]-leucine and [3H]-proline incorporation, respectively. Myocardial infarction (MI) was induced 4 weeks after 5/6th subtotal nephrectomy (STNx), following treatment with imidapril (Imid, 10 mg/kg daily) for 8 weeks. At endpoint, cardiac and renal function and structure were examined. Gene expression and Western Blot analysis were performed. Results: Only PDGF AB and BB stimulated NCM hypertrophy and NCF collagen syntheses, and increased hypertrophic, fibrotic and PDGFR gene expression (Fig). PDGF AB and BB activated p38MAPK and Akt, which were inhibited by their selective inhibitors. Imid treatment significantly improved cardiac and renal function, and reduced hypertrophic and fibrotic gene expression, post-STNx+MI. Imid treatment also reversed PDGFRβ and PDGF BB gene expression post-STNx+MI (Fig). Conclusion: The results demonstrated that only PDGF BB and AB via PDGFRβ subtype activation are involved in cardiac fibrosis in vitro and in vivo, suggesting the therapeutic potential of PDGFRβ subtype specific blockade in cardiac fibrosis.

Pathogenetic and Prognostic Role of Growth Factors in the Development of Chronic Heart Failure

To study the role of growth factors ((vascular endothelial growth factor (VEGF), platelet derived growth factor AB (PDGF-AB) and basic fibroblast growth factor (FGF-basic)) in the development and progression of chronic heart failure (CHF) in patients with ishcemic heart disease (IHD). We included in this study 94 patients with CHF. The control group comprised 32 persons. Blood serum levels of growth factors were determined at baseline and after 12 months of observation by enzyme-linked immunosorbent assay. VEGF, PDGF-AB and FGF-basic play an important role in the pathogenesis and progression of heart failure in patients with IHD, determining the increased risk of adverse cardiovascular events in this pathology. Serum activity of growth factors characterizes the severity and course of CHF: with disease progression levels of VEGF and FGF-basic decrease and PDGF-AB concentration increases. Initial low level of VEGF expression regardless of the sex of the patient's sex, significantly low level of FGF-basic and significantly high PDGF-AB in men characterizes unfavorable course of CHF. A correlation has been established between blood serum levels of VEGF, PDGF-AB and FGF-basic and severity and course of CHF.

Meloxicam and diclofenac do not change VEGF and PDGF-ABserum levels of platelet-rich plasma.

Platelet-rich plasma (PRP) application has gained widespread interest for musculoskeletal injuries. Nonsteroidal antiinflammatory drugs are frequently used in sports medicine before and/or after PRP application. Our study seeks to determine whether serum levels of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) levels of PRP would be affected by nonsteroidal antiinflammatory drugs. Two different final concentrations of diclofenac (0.5 μg mL-1 and 2.5 μg mL-1), meloxicam (0.8 μg mL-1 and 2.0 μg mL-1), and acetylsalicylic acid (final concentration 450 μm) were obtained in separate tubes with PRPs prepared from 20 healthy male volunteers. Medicine-free PRP was the control group. Growth factors were measured using ELISA. PDGF-AB and VEGF serum levels did not change with diclofenac, meloxicam, or acetylsalicylic acid addition. PDGF-AB and VEGF serum levels correlated with each other. Diclofenac, meloxicam, and acetylsalicylic acid did not affect PDGF-AB and VEGF serum levels.

Platelet-Rich Plasma Powder: A New Preparation Method for the Standardization of Growth Factor Concentrations

Background: Platelet-rich plasma (PRP) is widely used in sports medicine. Available PRP preparations differ in white blood cell, platelet, and growth factor concentrations, making standardized research and clinical application challenging. Purpose: To characterize a newly standardized procedure for pooled PRP that provides defined growth factor concentrations. Study Design: Controlled laboratory study. Methods: A standardized growth factor preparation (lyophilized PRP powder) was prepared using 12 pooled platelet concentrates (PCs) derived from different donors via apheresis. Blood samples and commercially available PRP (SmartPrep-2) served as controls (n = 5). Baseline blood counts were analyzed. Additionally, single PCs (n = 5) were produced by standard platelet apheresis. The concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor AB (PDGF-AB), transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), interleukin (IL)–1α, IL-1β, and IL-1 receptor agonist (IL-1RA) were analyzed by enzyme-linked immunosorbent assay, and statistical analyses were performed using descriptive statistics, mean differences, 95% CIs, and P values (analysis of variance). Results: All growth factor preparation methods showed elevated concentrations of the growth factors VEGF, bFGF, PDGF-AB, and TGF-β1 compared with those of whole blood. Large interindividual differences were found in VEGF and bFGF concentrations. Respective values (mean ± SD in pg/mL) for whole blood, SmartPrep-2, PC, and PRP powder were as follows: VEGF (574 ± 147, 528 ± 233, 1087 ± 535, and 1722), bFGF (198 ± 164, 410 ± 259, 151 ± 99, and 542), PDGF-AB (2394 ± 451, 17,846 ± 3087, 18,461 ± 4455, and 23,023), and TGF-β1 (14,356 ± 4527, 77,533 ± 13,918, 68,582 ± 7388, and 87,495). IGF-1 was found in SmartPrep-2 (1539 ± 348 pg/mL). For PC (2266 ± 485 pg/mL), IGF-1 was measured at the same levels of whole blood (2317 ± 711 pg/mL) but was not detectable in PRP powder. IL-1α was detectable in whole blood (111 ± 35 pg/mL) and SmartPrep-2 (119 ± 44 pg/mL). Conclusion: Problems with PRP such as absent standardization, lack of consistency among studies, and black box dosage could be solved by using characterized PRP powder made by pooling and lyophilizing multiple PCs. The new PRP powder opens up new possibilities for PRP research as well as for the treatment of patients. Clinical Relevance: The preparation of pooled PRP by means of lyophilization may allow physicians to apply a defined amount of growth factors by using a defined amount of PRP powder. Moreover, PRP powder as a dry substance with no need for centrifugation could become ubiquitously available, thus saving time and staff resources in clinical practice. However, before transferring the results of this basic science study to clinical application, regulatory issues have to be cleared.

Functional assessment of autologous platelet-rich plasma (PRP) after long-term storage at −20 °C without any preservation agent

Background: Platelet-rich plasma (PRP) is increasingly being used in the treatment of chronic wounds, pathologies of the musculoskeletal system, and in cosmetic medicine; however, the preparation of platelet-rich plasma is both time-consuming and requires invasive intervention. Additional costs are introduced if special equipment is used during preparation. The aim of the present study is to test whether autologous platelet-rich plasma (PRP) preserves the feature of growth factor release when stored at −20 °C after preparation.Method: Autologous PRP concentrates were prepared using whole blood samples obtained from 20 healthy subjects and divided into three parts to form three groups. Epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet derived growth factor-AB (PDGF-AB), insulin-like growth factor 1 (IGF-1), transforming growth factor-beta (TGF-β), and P-Selectin levels were immediately analysed in the control group. The other groups were defined as the experimental groups and were stored at −20 °C and analysed on the 7th and the 14th days. The same growth factors were tested in the experimental groups.Results: The growth factors (EGF, VEGF, PDGF-AB, IGF-1, TGF-β) and P-selectin levels were significantly decreased in the autologous PRP samples stored at −20 °C compared to the control group.Conclusion: The growth factor levels on days 7 and 14 suggest that autologous PRP can be stored at −20 °C without preservative agents, although in vivo studies are required in order to evaluate the clinical efficacy of the detected growth factor levels.

Comparison of fibrin clots derived from peripheral blood and bone marrow

ABSTRACTBackground: Autologous fibrin clots derived from peripheral blood (pb-fibrin clot) and bone marrow (bm-fibrin clot) are thought to be effective for tissue regeneration. However, there is no report detailing the amount of growth factors in pb-/bm-fibrin clot. In this study we evaluated the amount of growth factors in human pb-/bm-fibrin clot, and prove the validity of fibrin clot for clinical use. Methods: Human pb-/bm-fibrin clots were obtained during surgery. In the first experiment, enzyme-linked immunosorbent assay (ELISA) was performed for detecting the amount of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-β), platelet derived-growth factors-AB (PDGF-AB), and stromal cell-derived factor-1 (SDF-1). In the second experiment, the efficacy of fibrin clot on the osteogenic differentiation and fibroblast proliferation was evaluated. Pb-/bm-fibrin clots were incubated in human osteoblast derived from mesenchymal stromal cells (MSCs) or human skin fibroblast. Alizarin red staining and real-time PCR (COL1A1, RUNX2) were performed for the detection of osteogenic potential. Cell-growth assay (WST-8) and real-time PCR (COL1A1) were also performed for the detection of the potential of fibroblast proliferation. Results: ELISA analysis revealed that the amount of VEGF, HGF, bFGF, IGF-1, and SDF-1 of bm-fibrin clot group is higher than that of pb-fibrin clot group with statistical differences. Besides, we confirmed that bm-fibrin clot has much potential for the osteogenic differentiation and fibroblast proliferation. Conclusion: The positive outcomes confirm the efficacy of pb-/bm-fibrin clot, and bm-fibrin clot was proved to have much potential for tissue regeneration compared with pb-fibrin clot. The current study showed the potential of a strategy for regenerative medicine using bm-fibrin clot.

Serum soluble CD40L concentration depending on the stage of multiple myeloma and its correlation with selected angiogenic cytokines.

INTRODUCTION Little is known about the CD40L-CD40 pathway in hematologic malignancies, especially in multiple myeloma (MM). OBJECTIVES The aim of the current study was to evaluate serum soluble CD40 ligand (sCD40L) concentrations in patients with newly diagnosed MM prior to treatment at different stages of disease, compared with healthy controls. To assess the clinical significance of sCD40L, we assessed correlations between the levels of sCD40L and those of angiogenic cytokines: interleukin 6 (IL-6), soluble receptor of IL-6 (sIL-6R), tumor necrosis factor α (TNF-α), soluble vascular cell adhesion molecule 1 (sVCAM-1), and platelet-derived growth factor AB (PDGF-AB), as well as with well-established biomarkers of MM activity (lactate dehydrogenase activity and percentage of bone marrow plasma cells) and with a marker of platelet activation (β-thromboglobulin). PATIENTS AND METHODS The study group consisted of 41 patients with newly diagnosed MM; the control group consisted of 30 healthy subjects. The level of sCD40L was determined using an enzyme-linked immunosorbent assay. RESULTS The level of sCD40L was significantly higher in patients with MM than in controls and increased with the stage of the disease. Moreover, it significantly correlated with the levels of IL-6, sIL-6R, sVCAM-1, PDGF-AB, as well as the levels of MM activity markers and β-thromboglobulin. CONCLUSIONS Our findings indicate that increased serum sCD40L levels may be related to angiogenesis in patients with MM. This protein has potential clinical usefulness in MM and may be considered as an additional prognostic marker. The correlation of sCD40L with β-thromboglobulin may indicate that in patients with MM sCD40L derives from activated platelets.

Plasma rich in growth factors stimulates proliferation, migration, and gene expression associated with bone formation in human dental follicle cells

Background/purposePlasma rich in growth factors (PRGFs), which is prepared from autologous blood from patients, has been reported with regards to bone regeneration for dental implants. Human dental follicle cells (hDFCs) have the capacity to commit to multiple cell types such as the osteoblastic lineage. The aim of this study is to evaluate the effects of PRGFs for mineralization in hDFCs. Materials and methodsPRGFs was prepared from whole blood centrifuged at 460g for 8 minutes. hDFCs isolated from the dental follicle with collagenase/dispase were cultured with growth medium or osteogenic induction medium (OIM) containing PRGFs or fetal bovine serum. Concentrations of the growth factors were examined using an enzyme-linked immunosorbent assay kit. A cell migration assay was used for two-dimensional movement. Gene expressions were examined with real-time polymerase chain reaction using a DyNAmo SYBR Green quantitative polymerase chain reaction kit. ResultsThe platelet concentration in PRGF Fraction 2 was 2.14-fold higher than in whole blood. White blood cells were not detected in PRGFs. Transforming growth factor-β levels were higher than insulin-like growth factor-1, platelet-derived growth factor-AB and -BB, and vascular endothelial growth factors in PRGF Fraction 2. Proliferation and migration by hDFCs increased in OIM supplemented with PRGFs in a dose-dependent manner and were higher in hDFCs cultured in OIM plus 10% PRGFs compared with OIM plus 10% fetal bovine serum. PRGFs upregulated the gene expression of type I collagen, osteomodulin, alkaline phosphatase, bone morphogenic protein-4, and transforming growth factor-β in hDFCs. ConclusionPRGFs may promote bone regeneration due to it including high levels of growth factors.

Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Sponge-Like Dressings Based on the Association of Chitosan and Sericin for the Treatment of Chronic Skin Ulcers. II. Loading of the Hemoderivative Platelet Lysate

Platelet lysate (PL) was loaded into dressings based on chitosan glutamate (CSG) low and high molecular weight, sericin (Ser), and glycine (Gly). A synergic effect of Ser and PL on fibroblast proliferation was proved in vitro. Two different PL loading approaches were considered: the first provided to prepare dressings by freeze-drying a mixture of PL and CSG/Gly/Ser solution, the second approach consisted in the extemporarily loading of PL in the CSG/Gly/Ser freeze-dried dressings. As for the first approach, PL loading did not produce any variation in dressing mechanical properties. Such dressings absorbed a high amount (about 8-fold of dry weight) of phosphate-buffered saline (fluid mimicking wound exudate), forming a gel with pseudoplastic and elastic properties. Platelet-derived growth factor AB assay indicated that neither freeze-drying nor the excipients alter PL growth factor content. As for the second approach, mechanical and rheological properties of the gel formed upon PL absorption enabled to choose a PL loading of about 90 μL/cm2. Upon contact with fibroblasts, all PL loaded formulations increased the number not only of viable cells but also of those in the proliferative phase. Histological studies effected on human skin strips pointed out the positive effect of PL loaded dressings on dermal matrix reconstruction.

Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time

IntroductionPlatelet derived biomaterials represent a key source of cytokines and growth factors extensively used for tissue regeneration; wound healing and tissue repair. Our study was to quantify platelets and growth factors released by PRP when prepared at different centrifugal force (g) and time. Material and MethodsOur study was approved by the institutional ethical committee. One hundred millilitres of whole blood (WB) was collected in bag with CPDA as the anticoagulant(AC); (14 mL for 100 mL WB ratio). Nine aliquots of 10 mL each were made from the bag and set of three aliquots were made a group. PRP was prepared at varying centrifugal force (group A: −110 g, group B: −208 g & group C: −440 g ) & time (1: −5 min, 2: −10 min & 3: −20 min). Contents of each PRP prepared were analysed. Commercial sandwich ELISA kits were used to quantify the concentrations of CD62P (Diaclone SAS; France), Platelet derived growth factors-AB (Qayee-Bio; China), transforming growth factor-β1 (DRG; Germany) and vascular endothelial growth factor (Boster Immuno Leader; USA) released in each PRP prepared. ResultsEight volunteers were enrolled in the study (24–30 years). The baseline blood counts of all the volunteers were comparable (p ≥ 0.05). Mean ± SD of platelet yield of all nine groups ranged from 17.2 ± 4.2% to 78.7 ± 5.7%. Each PRP was activated with calcified thromboplastin to quantify the growth factors released by them. Significantly higher (p < 0.05) transforming growth factor-β1 and vascular endothelial growth factor were released compared to the baseline. ConclusionOur study highlights the variation in both force (g) and time results in changes at cellular level and growth factor concentrations.